Methods for use of apoptotic cells to deliver antigen to dendritic cells for induction or tolerization of T cells

ABSTRACT

This invention relates to methods and compositions useful for delivering antigens to dendritic cells which are then useful for inducing antigen-specific cytotoxic T lymphocytes and T helper cells. This invention also provides assays for evaluating the activity of cytotoxic T lymphocytes. According to the invention, antigens are targeted to dendritic cells by apoptotic cells which may also be modified to express non-native antigens for presentation to the dendritic cells. The dendritic cells which are primed by the apoptotic cells are capable of processing and presenting the processed antigen and inducing cytotoxic T lymphocyte activity or may also be used in vaccine therapies.

This application claims priority to U.S. provisional applications Ser.No. 60/075,356 filed Feb. 20, 1998, Ser. No. 60/077,095 filed Mar. 6,1998; and Ser. No. 60/101,749 filed Sep. 24, 1998 all of which areincorporated herein by reference.

This invention was made with United States Government support underNational Institutes of Health grant AI-39516, AI-13013 and AI 39672. TheUnited States Government has certain rights in this invention.

FIELD OF THE INVENTION

This invention relates to targeted antigen presentation in the immunesystem. In particular, this invention relates to the use of apoptoticcells to deliver antigens to dendritic cells for induction ortolerization of antigen-specific T cell responses. The apoptoticcell-mediated antigen delivery system described herein has a wide rangeof preventive, diagnostic and therapeutic applications.

BACKGROUND OF THE INVENTION

CD8⁺ cytotoxic T lymphocytes (CTLs) play a critical role in immunedefense against infectious agents, tumors and transplants. ClassI-restricted CD8⁺ T cells have been implicated in the recognition anddestruction of such clinically important targets as HIV-1 (1-3),Influenza A (4), malaria (5), cytomegalovirus infected cells (6),Epstein-Barr virus (7), and human melanoma cells (8-9). Therefore,establishing methods for inducing and expanding populations ofantigen-specific CD8⁺ CTLs remains an important objective in thedevelopment of therapeutic treatments against infectious disease andcancer.

CD8⁺ CTLs are activated by antigens which have been processed andpresented on major histocompatibility (MHC) class I molecules on thesurface of specialized antigen presenting cells. A number of antigenpresenting cells have been identified which activate T cells includingmacrophages/monocytes, B cells and bone marrow derived dendritic cells.Of these, dendritic cells are recognized as playing a pivotal role inthe initiation of CD8⁺ CTL responses (10).

An important feature of dendritic cells is their ability to efficientlyprocess and present antigens on MHC class I and/or class II molecules.Depending on the antigen processing pathway, dendritic cells are capableof activating distinct populations of CTLs. In the case of influenzavirus, for example, it is known that the class I pathway for inducingCD8⁺ CTLs requires adequate delivery of infectious viral antigen intothe cytoplasm, whereas the purely endocytic pathway deliversnoninfectious virions for presentation only to CD4⁺ T helper cells (U.S.Ser. No. 08/282,966). Thus, although dendritic cells efficientlyactivate class I-restricted CTLs, access to the MHC class I pathway forinduction of CD8⁺ T cells normally requires endogenous synthesis ofantigen. Accordingly, it is important to identify antigen deliverysystems which efficiently mediate access of exogenous antigen to the MHCclass I-restricted antigen presentation pathway in order to generateantigen-specific CD8⁺ T cell responses.

Recently, a number of approaches have been reported for delivery ofexogenous antigen to the MHC I processing pathway of dendritic cells.These methods include coupling antigens to potent adjuvants (11-15),osmotic lysis of pinosomes after pinocytic uptake of soluble antigen(16), or insertion of antigen in pH-sensitive liposomes (17). However,these prior approaches still pose a number of limitations on thedevelopment of effective therapeutic treatments. For example, Nair etal. report that dendritic cells do not efficiently internalizeantigen-containing liposomes in vivo (18). Further, osmotic lysis ofdendritic cell pinocytic vesicles may be difficult to perform in vivo,and may result in inefficient antigen delivery to the MHC class Iprocessing compartment. Also, the use of powerful adjuvants may beundesirable for some clinical applications.

Pulsing dendritic cells directly with exogenous antigen using wholecells in viable or irradiated forms, membrane preparations, or antigenspurified from natural sources or expressed as recombinant products hasalso been previously reported (WO 94/02156). These prior methods,however, do not recognize forms of cell death or the processing pathwaysantigens from dead or dying cells access in the dendritic cell system.

Rubartelli et al. report that dendritic cells, unlike macrophages, failto take up opsohized particles or necrotic cells in vitro, but canefficiently engulf cells undergoing apoptotic programmed cell death(19). The mechanism of internalization of apoptotic cells by dendriticcells, however, is different than in macrophages, indicating thatresults in the macrophage system are not necessarily predictive ofdendritic cell responses. In addition, Rubartelli et al. does not showpresentation of engulfed material and therefore speculates as to thefate of such material.

SUMMARY OF THE INVENTION

This invention provides highly efficient methods for deliveringexogenous antigens to dendritic cells and inducing antigen-specific Tcell activation. In particular, the methods described in this inventionare directed toward developing therapies for increasing patient immunityto chronic infections and tumors by 1) inducing tumor or infected cellsto undergo apoptosis, 2) having the apoptotic tumor or infected cellsgain access to phagocytic, maturing dendritic cells, and 3) exposing theapoptotic cell-primed dendritic cells expressing antigen of interest toT cells, in vivo or in vitro, for induction of antigen-specific T cellresponses.

This invention further provides that the population of donor cellsexpressing said antigen can be induced to undergo apoptosis using avariety of methods including, but not limited to, viral infection,irradiation with ultraviolet light, gamma radiation, cytokine treatment,or depriving donor cells of nutrients in the cell culture medium. It isalso contemplated by this invention that the dendritic cells can beexposed to a preparation of donor apoptotic cell fragments, blebs orbodies rather than whole apoptotic cells.

In another embodiment of this invention, the donor cells can betransfected, transduced or transformed to express foreign antigens priorto induction of apoptosis. A variety of such antigens may be expressedby the donor cells including, but not limited to, viral antigens, tumorantigens, toxins, microbial antigens, and autoimmune antigens.

Accordingly, this invention also provides a method of generatingantigen-specific cytotoxic T lymphocytes comprising providing apopulation of apoptotic cells, or membrane containing fragments thereof,expressing said antigen, exposing dendritic cells to said apoptoticcells for a time sufficient to allow said antigen to be internalized andprocessed by the dendritic cells, and exposing T lymphocytes in vivo tosaid dendritic cells for a time sufficient to induce said lymphocytes tobecome antigen-specific T lymphocytes. This invention furthercontemplates induction of antigen-specific T lymphocytes in vitro.

In another embodiment of this invention apoptotic cells expressing anantigen to be presented by dendritic cells are administered to anindividual in an amount and in a location so as to prime dendritic cellsin vivo.

Methods of preventing and treating disease are also provided by thisinvention which comprises administering to an individual in need oftreatment, a therapeutically effective amount of dendritic cells whichhave been primed by apoptotic cells.

In addition to the methods of this invention, this invention alsoprovides dendritic cells which have been primed by apoptotic cells, andin particular, human dendritic cells which are prepared according to themethods of this invention. This invention further provides transformedapoptotic cells, or a preparation of transformed apoptotic cellfragments, and in particular, human apoptotic cells which are preparedaccording to the methods of this invention.

In another embodiment of this invention the apoptotic cell deliverysystem is reconstituted in vitro using liposomes comprising antigen andapoptotic cell proteins, factors or ligands which enhance uptake andprocessing of antigen in dendritic cells.

Based on the results disclosed herein, one may include in a liposomeligands for integrin receptors, including, but not limited to theα_(v)β₅ and α_(v)β₃ integrin receptors for enhancing uptake andprocessing of antigens in dendritic cells. An example of such a ligandis lactadherin. Other examples of proteins which may enhance uptake ofapoptotic particles include thrombospondin. Similarly, ligands whichbind CD36 may also be used. In a preferred embodiment ligands for bothintegrin and CD36 would be used.

Heat shock proteins (HSP) may also be included to facilitate antigenuptake by dendritic cells. For example, HSP 70 family members,especially HSP 70, and HSP 90 members, especially HSP 84 and GP 96 maybe used. Certain antigens obtained from infectious sources may alreadybe bound to HSPs. Alternatively, antigens may be complexed to HSP exvivo.

The identification of the α_(v)β₅ integrin receptor as mediating antigenuptake by dendritic cells provides one with a means of modulating thatantigen uptake by either increasing or decreasing the activity of theα_(v)β₅ receptor. Accordingly, this invention provides means forenhancing antigen uptake and processing by dendritic cells in vitro orin vivo, by adding an α_(v)β₅ ligand to the dendritic cells inconjunction with the antigen to be taken up and processed.Alternatively, one could attenuate the antigen uptake activity of thedendritic cells by blocking the α_(v)β₅ receptor, or other receptorswhich are involved in medrating antigen uptake by the dendritic cells.

This invention also includes pharmaceutical compositions for chargingdendritic cells comprising a ligand which activates antigen uptake, suchas an α_(v)β₅ ligand, with or without an antigen to be processed andpresented by dendritic cells. As used in this invention such ligands maybe considered to function as immunoadjuvants.

This invention also provides methods for modulating T cell responses invivo or in vitro to induce immunity or tolerance by controling DCmaturation.

In one embodiment, T cell immunity can be induced when immature DCs arecaused to mature by the addition of appropriate maturation factors afterthe uptake of apoptotic particles. Examples of maturation factors thatcan be used in this invention include monocyte conditioned medium,TNF-α, IL-1β, IL6, PGE₂, IFN-α, CD40 ligand, and necrotic cells.

In another embodiment, tolerance can be induced to self or foreignantigens by maintaining DCs in their immature state in the absence of amaturation signal.

It is a general object of this invention to provide a method of usingapoptotic cells, or apoptotic cell fragments, to efficiently deliverspecific antigens to dendritic cells which then process and present saidprocessed antigens on their surface for stimulation or tolerization ofcytotoxic T lymphocytes.

It is another object of this invention to generate antigen-specificcytotoxic T lymphocytes either in vivo or in vitro by using thedendritic cells generated by the methods described herein.

It is also an object of this invention to provide a method ofprophylactic or therapeutic treatment for a variety of cancers,autoimmune diseases, and pathogens using the dendritic cells describedherein.

DESCRIPTION OF FIGURES

FIGS. 1a, 1 b and 1 c: Dendritic cells acquire antigen frominfluenza-infected cells and induce class I-restricted CTLs. FIGS. 1a, 1b. Varying doses of influenza-infected syngeneic monocytes [FIG. 1a] andallogeneic HLA-A2.1⁻ monocytes were co-cultured with DCs and T cells for7 days. After 8 to 10 hours, 10⁶ T-cells and 3.3×10⁴ DCs derived fromHLA-A2.1⁺ were added. On day 7, cytolytic activity was tested usingsyngeneic influenza-infected macrophages [FIG. 1a] or T2 cells, aHLA-A2.1⁺ cell line which lacks the transporters of antigen processingor TAP, pulsed with the immunodominant influenza matrix peptide (35,36)[FIG. 1b] as targets. Influenza-infected DCs served as a control in allexperiments in order to measure the donor's CTL responsiveness toinfluenza. Responses varied as a function of the individual's priorexposure to influenza. Background lysis ranged from 0-5% for theuninfected monocytes and 0-20% for the unpulsed T2 cells. FIG. 1c.Uninfected syngeneic DCs were co-cultured with influenza-infectedallogeneic monocytes for 2 days prior to being used as targets for CTLs.Control targets included influenza-infected syngeneic DCs andinfluenza-infected allogeneic monocytes. Effector: target ratio=45:1.These results in FIGS. 1a, 1 b and 1 c are representative of 8experiments and the values shown represent the mean from triplicatewells.

FIGS. 2a and 2 b: Antigen transfer is not due to live influenza virus orfree peptide. [FIG. 2a]. 5×10⁴infected allogeneic HLA-A2.1⁻ monocyteswere cultured for 10 hours, after which the media from the wellscontaining infected monocytes was removed and added to fresh wellscontaining HLA-A2.1⁺ T-cells and DCs [10 hr transfer]. Media from theinfected monocyte was also passed through a 0.45 micron filter, prior toaddition to wells containing HLA-A2.1⁺1 T cells and DCs [filter].Effector:target ration=30:1. [FIG. 2b]. 5×10³ infected allogeneic(HLA-A2.1 mismatched) monocytes were cultured for 10 hours, after whichT-cells and DCs were added to the wells [filled squares]. Alternatively,the medium from the wells containing infected monocytes (HLA-A2.1mismatched) was removed and transferred to fresh wells containingT-cells and DCs [stars]. Other cultures were established in which themedium was first spun at 250×g in a GH-3.8 Beckman Rotor for 10 minutes.The resulting supernatant fraction [up triangle] versus the pellet [downtriangle] was added to the T cell and DC containing cultures. After 7days, cytolytic activities in the T cell populations were determined. T2cells pulsed with the influenza matrix peptide were used as targets.These results are representative of 3 experiments and the values shownrepresent the mean from triplicate wells.

FIGS. 3a, 3 b, 3 c and 3 d: Apoptosis is required for delivery ofantigen to DCs. [FIG. 3a]. Monocytes were infected with live andheat-inactivated influenza virus. After overnight culture, cells werestained with annexin V-FITC [ann V] and propidium iodide [PI], andanalyzed by flow cytofluoremetry (FACscan®). Early apoptotic cells aredefined by the ann V⁺, PI⁻ population (37). [FIG. 3b]. AllogeneicHLA-A2.1⁻ monocytes were infected with either heat-inactivated,non-replicating influenza virus (15) or with live influenza virus. 5×10³monocytes were cultured for 10 hours, after which the media from thewells containing the infected monocytes was removed and added to freshwells containing T cells and uninfected DCs [10 transfer].Alternatively, the infected monocytes were co-cultured with the T cellsand DCs [co-culture]. Heat-inactivated influenza and liveinfluenza-infected DCs 1 served as the positive controls for theexperiment. After 7 days, T cells were tested for cytolytic activityusing T2 cells as targets. [FIG. 3c]. Apoptosis of influenza-infectedmonocytes was inhibited using Z-VAD-CHO, an irreversiblepeptide-aldehyde inhibitor of caspases, the enzymes that are involved inthe apoptotic pathway. 1×10⁴ infected monocytes were exposed to varyingdoses of Z-VAD and cultured for 10 hours. The media from these wells wasthen transferred to fresh wells containing 2×10⁵ T cells and 6.67×10³DCs. Cytolytic activity was determined as in [FIG. 3b]. [FIG. 3d]. Cellsof the human kidney epithelial 293 cell line (ATCC), were infected withinfluenza virus, and cultured for 10 hours at 37° C. Apoptosis was theninduced by exposure to 60 mJ/cm² of UVB irradiation. Necrosis wasachieved by incubating the 293 cells in a hypotonic solution for 30minutes at 37° C., as determined by incorporation of trypan blue.Apoptotic or necrotic 293 cells were co-cultured with T cells anduninfected DCs for 7 days. Cytolytic activity was determined as in [FIG.3b]. Uninfected 293 cells cultured with T cells and DCs failed to inducevirus-specific CTLs [data not shown]. These results in FIGS. 3a-3 d arerepresentative of 9 experiments and the values shown represent the meanfrom duplicate or triplicate wells.

FIGS. 4a and 4 b: Generation of CD8+ CTLs requires CD4+ T-cell help anddendritic cells. [FIG. 4a]. 5×10⁴ influenza-infected allogeneicmonocytes were co-cultured with DCs and CD8⁺ T cells. Highly purifiedCD8⁺ T cells (13) were cultured either alone, 50 U/ml human IL-2 or withCD4⁺ T cells. IL-2 was added on days 0 and 3. The ratio of CD8⁺: CD4⁺ Tcells was 1:3, matching the ratio in peripheral blood. Cytolyticactivities were determined using T2 cells pulsed with the influenzamatrix peptide as targets. Effector: target ratio=10:1. This data isrepresentative of 3 experiments, each testing various doses of apoptoticmonocytes. [FIG. 4b]. Influenza-infected syngeneic monocytes wereco-cultured with various APCs and bulk T cells. Uninfected DCs,uninfected monocytes and mixtures of both were used as the APCs.Infected monocytes alone and infected DCs served as controls for theexperiment. Cytolytic responses were measured as described. Effector:target ratio=30:1.

FIGS. 5a, 5 b and 5 c: Dendritic cells engulf apoptotic monocytes.Influenza-infected monocytes and uninfected DCs were co-cultured for upto 10 hours. [FIG. 5a]. Cells were stained by immunofluorescence withanti-CD8 [isotype control] or anti-p55 followed by goat anti-mouse-FITCand incubation with 4′6′ diamidino-2-phenylindole (DAPI) [Sigma]. Thenucleus of the DCs [arrows] are lobulated and euchromatic as comparedwith the pyknotic, fragmented nucleus of apoptotic cells [open arrows].DAPI⁺ material from an apoptotic cell appears to be within the cytoplasmof a p55⁺ DC. [FIG. 5b]. Electron microscopy revealed apoptotic materialand apoptotic cells [AC] within the cytoplasm of glutaraldehyde fixedDCs (38). [FIG. 5c]: DCs are identified by immuno-electron microscopywith anti-CD83 visualized by 10 nm gold beads. Inset 1 shows thelocalization of CD83 [arrows] on the processes of the DC. Note thedouble membrane in inset 2 [arrowheads], which reveals an apparentlyintact plasma membrane of the cell. After immuno-labeling theparaformaldehyde fixed DCs, the cell pellet was augmented withglutaraldehyde fixed tumor cells [*].

FIG. 6: Apoptotic transfected 293 cells serve as antigenic material for‘cross-priming’ of CD8⁺ T cells.

FIG. 7: CD8⁺ T-cells and not CD4⁺ cells are responsible forinfluenza-specific cytotoxicity.

FIG. 8: Immature DCs phagocytose Influenza Infected EL4 cells andstimulate Influenza-specific CTLs.

FIGS. 9A and 9B: Immature DCs, but not mature DCs or macrophagescross-present antigenic material from apoptotic cells and become targetsof antigen-specific CTLs. [FIG. 9a]. Various populations of HLA-A2.1+professional antigen presenting cells (APCs) were co-cultured withHLA-A2.1− influenza infected monocytes. [FIG. 9b]. Influenza infectedCD83⁺ DCs serve as better targets in a CTL than infected CD83− DCs orCD14⁺ macrophages. Controls included infected and uninfected APCs.HLA-A2.1 monocytes were also tested as targets to demonstrate theabsence of lysis when using a mis-matched target. Effector: TargetRatios=45:1 and 15:1.

FIGS. 10A, 10B, 10C, 10D and 10E: Development of MHC-peptide complexesconsisting of a B cell derived I-Eα peptide and DC-derived, I-A^(b) MHCII products.

[FIG. 10A]: C57BL/6, I-1^(b) DCs were cultured 20 hrs with no peptide,with 10 μM preprocessed peptide, or with 2×10⁶ B blasts from H-2d BALB/C[I-E⁺] or H-2b C57BL/6 [I-E⁻] mice. The cultures were labeled withbiotin Y-Ae antibody that recognizes the complex of I-Ab and I-E peptide[y-axis] and CD86 costimulator. The CD86-rich DCs [black arrows] acquireI-E peptide. No labeling is seen with an IgG2b isotype control to Y-Ae[bottom row].

[FIG. 10B]: As in FIG. 10a, but DC-BALB/C blast co-cultures [top] or DCsonly [bottom] were labeled with other mAbs to show that CD11c⁺ I-Ab⁺I-Ad⁻ DCs selectively acquired Y-Ae [arrows].

[FIG. 10C]: As in FIG. 10a, C57BL/6 DCs were cultured for the indicatedtimes with different doses of I-Eα peptide or 2×10⁶ B blasts from BALB/Cmice and double labeled for Y-Ae and CD86. Y-Ae signals [MeanFluorescence Index] are shown for CD86⁺ mature DCs.

[FIG. 10D]: As in FIG. 10a, but graded doses of B blasts [4 days LPSstimulation] and small B cells were compared as a source of I-Eαpeptide.

[FIG. 10E]: As in FIG. 10a, but the DCs were sorted from the 20 hrcultures of DCs with peptide or B blasts and used to stimulate IL-2production [3H-thymidine uptake] from a T-T hybridoma specific for acomparable epitope to the Y-Ae monoclonals.

FIGS. 11A, 11B, 11C and 11D: Antigen transfer requires cellularprocessing and occurs from xenogenic cells.

[FIG. 11A]: To rule out transfer of peptide from B cells to DCs, Bblasts were separated from immature DCs [6 day mouse bone marrowcultures] by a Transwell filter or added to mature DCs, isolated assingle nonadherent cells from day 8 mouse marrow cultures [methods].

[FIG. 11B]: Y-Ae epitope formation is blocked by NH₄Cl. DCs werecultured 20 h with B blasts with or without NH₄Cl at 5-20 mM. Thecultures were stained for Y-Ae and CD86.

[FIG. 11C]: EBV-transformed human B cell lines [0,0.5 or 5 cells/DC;frequency plots, top] or human monocytes [3 cells/DC; dot blots bottom]serve as a source of I-Eα peptide. The monocytes were untreated, subjectto freeze thawing, or induced to apoptose by infection with influenza[monocytes-flu] or by UV light [not shown].

[FIG. 11D]: Development of MHC-peptide complexes consisting of a Bcell-derived I-Eα peptide and DC-derived, I-A^(b) MHC II products.EBV-transformed human B cells lines (3 cells/DC; necrotic and apoptotic)served as a source of I-Eα peptide for mouse DCs. An isotype-matchedIgG2b antibody was used as a control for the specific Y-AE labeling thatdeveloped on the CD86-rich DCs.

FIGS. 12A, 12B and 12C: Antigen transfer is preceded by phagocytosis.(A) Top, the formation of Y-Ae after 20 h of DC-B blast co-culture withor without 20 mM NH₄Cl. The DCs then were sorted as double positive forPE-CD11c and FITC-CD86, or in a second experiment, just FITC-CD86⁺, andcultured for 12 h without NH₄CL (bottom). Y-AE (biotin Y-Ae followed byCychrome-Avidin) quickly regenerated in DCs that had been blocked byNH₄Cl. No Y-Ae was found in saponin-permeabilized samples ofNH₄CL-treated cultures. (B) Immature DCs were cultured with or without20 mM NH₄Cl and 10 μM preprocessed I-E peptide for 15 h, and then Y-Aelevels were measured on the FACS®. CD86 (not shown) and Y-Ae epitopeformed in the presence of NH₄Cl.

[FIG. 12C].Same as FIG. 12A, but the sorted DCs were used to stimulateIL-2 release from a T-T hybrid specific for the I-A^(b)/Ep complex [IL-2monitored by DNA synthesis in test T blasts]. If the DCs were culturedwith B blasts in the absence of NH₄Cl [ ], there is strong T cellstimulation, even if the DCs were fixed. However, if the DCs werecultured with B blasts and NH₄Cl [ ], then there is regeneration of theT cell epitope if the DCs are not fixed prior to the 24 hr culture withT cells [compare left and right].

FIGS. 13A and 13B: Antigen transfer to DCs in vivo.

[FIG. 13A]: 2×10⁶ mature, marrow-derived, H-2d DCs were injected intothe foot pads of H-2b mice. 2 days later, sections of the poplitealnodes were stained for Y-Ae [blue] and B220 B cell marker [brown] andphotographed at low and high power [50 and 200×]. Blue dendriticprofiles were scattered throughout the T cell areas.

[FIG. 13B]: As in FIG. 13A, but cell preparations enriched in DCs wereexamined by FACS. Several mouse strains were tested as donors andrecipients of the marrow-derived DCs. F1 mice [left column] were used asa positive control. The cells were double labeled for Y-Ae or isotypematched control mAb [y-axis] and for different markers [x-axis]. Y-Ae⁺DCs [black] and B cells [white] are arrowed.

[FIG. 13C]: As in B, but the DCs were from B6.I-E transfenic (right) orB6 mice (left). Processing by host CD11c⁺ DCs to form the Y-Ae epitopeoccurs when the donor and recipient DCs differ only in terms of I-Eexpression.

FIGS. 14A and 14B: Immature but not mature dendritic cells efficientlyphagocytose apoptotic cells.

Freshly isolated, blood monocytes were infected with live influenza A,PR/8 [Spafas Inc.], labeled with the PKH26-GL fluorescent cell linkercompound [Sigma Biosciences], and incubated at 37° C. for 6-8 hoursallowing apoptosis to occur. Macrophages, immature DCs and mature DCswere dyed with PKH67-GL and added to the culture wells containing theapoptotic monocytes at a ratio of 1:1. Cells were analyzed by FACScan®where double positive cells indicate uptake of the apoptotic cells bythe various APCs [panels iii., vi., ix]. We used the various APCs aloneto establish the proper settings [panels i., iv., vii.] Note, as theforward scatter of the APCs increased, the dying monocytes were excludedfrom the established region [panels ii., v., viii.]. After 2 hr., 80% ofthe macrophages 50% of the immature DCs, and less than 10% if the matureDCs had engulfed the apoptotic monocytes [A]. In an independentexperiment, macrophages [squares], immature DCs [diamonds] and matureDCs [circles] were prepared and co-cultures with apoptotic monocyteswere established as described above, and FACS® was performed at varioustime points. Percent phagocytosis was calculated based on the number ofdouble positive cells [B].

FIG. 15: Low temperature, Cytochalasin D and ETDA block phagocytosis ofapoptotic cells by immature DCs.

Apoptotic monocytes and immature DCs were prepared as described.Immature DCs were pre-incubated at 4° C. [A], in the presence of varyingconcentrations of Cytochalasin D [B], or EDTA [C] for 30 minutes.Apoptotic monocytes were then added to the DC cultures at 4° C. [A] or37° C. [B,C]. FACS® analysis was performed after 1-2 hrs. Data shown inFIG. 2 are representative of 5 independent experiments in whichinfluenza infected monocytes or UVB irradiated HeLa cells were sourcesof apoptotic food for the immature DCs. Percent inhibition+/−standarddeviation for these experiments were: 4° C., 85%+/−7%; 10 μMCytochalasin D, 69%+/−3%; and 2 mM EDTA, 76%+/−14%.

FIG. 16: Immature DCs engulf influenza infected monocytes.

Influenza infected apoptotic monocytes were co-cultured with immatureDCs for 1 hr after which the cells were adhered to a cover slip andfixed with acetone. Immunofluorescence was performed with anti-influenzanucleoprotein antibodies [NP] and Texas red conjugated goat anti-mouseIgG; and biotinylated anti-HLA-DR [DR] followed by FITC conjugatedstreptavidin. Large arrowhead indicates apoptotic cell outside the DCprior to engulfment. Small arrows indicate apoptotic material derivedfrom the influenza infected monocytes within DR⁺ vesicles of the DC.These image were not generated on a confocal scope, so the structures ofthe DC underlying the apoptotic cell can be seen.

FIGS. 17A and 17B: Immature DCs but not mature DCs nor macrophagescross-present antigenic material derived from apoptotic cells.

Various populations of HLA-A2.1⁺ antigen presenting cells [APCs] wereco-cultured with HLA-A2.1⁻ influenza infected monocytes. After 12 hrs,the APCs were loaded with ⁵¹Cr and used as targets for HLA-A2.1⁺influenza-reactive CTL lines. Mature DCs were isolated by labeling withthe DC-restricted marker CD83, followed by cell sorting on the FACSort®[Becton Dickinson]. Immature DCs were CD14⁻ and sorted by FACSort® as aCD83⁻ population. Mature macrophages were generated by culturing anadherent mononuclear cell fraction in a Teflon beaker for 9 days.Effector: Target Ratios=45:1 and 15:1 [A]. Controls included infectedand uninfected mature DCs, immature DCs and macrophages. The HLA-A2.1⁻monocytes used as a source of apoptotic material were also tested astargets to demonstrate the absence of lysis when using a mis-matchedtarget. Effector: Target Ratios=45:1 and 15:1. Results arerepresentative of 3 experiments and the values shown represent the meanof triplicate wells [B].

FIG. 18: Intracellular but not extracellular CD83 expressiondistinguishes immature DCs from mature DCs and macrophages.

Macrophages [A], immature DCs [B] and mature DCs [C] were prepared aspreviously described. Cells were incubated with anti-CD83, a DCmaturation marker, either untreated or post-saponin treatment. Thelatter permeablized the cells allowing for intracellular staining. Cellswere then labeled using a PE-conjugated GAM-Ig [Biosciences] A controlisotype matched antibody was used.

FIGS. 19A, 19B, and 19C: Protein and mRNA expression of α_(v)β₅ and CD36are down regulated during DC maturation.

Immature DCs [A] and mature DCs [B] were incubated with anti-α_(v)β₃[clone 23C6, Pharmingen], anti-CD36 [clone FA6, obtained from the Vthinternational workshop on leukocyte differentiation antigens], oranti-α_(v)β₅ [clone P1F6, Chemicon], followed by PE-conjugated GAM-Ig[Biosciences]. All cells were analyzed by FACScan®. [C] RNA was purifiedfrom highly purified sorted cell populations of immature and mature DCsas previously described. RT-PCR was carried out and after 30 cycles ofPCR the distinct bands for β₃, β₅ and CD36 could be seen in the immatureDCs [lane 1]. In mature DCs only a faint band for CD36 and no band forβ₅ could be visualized [lane 2] indicating minimal mRNA. In contrast, aband was evident for β₃ in the mature DCs. Note, the doublet for β₃ isan artifact in this particular exposure and does not indicate two uniquebands [lane 2]. As a positive control, extracts from Bowes melanomacells were run, which are known to express β₃ and CD36 (29) [lane 3]. Asnegative control, the Bowes melanoma cells were run in the absence of areverse transcriptase [lane 4].

FIGS. 20A and 20B: Direct inhibition of phagocytosis by anti-α_(v)β₅ andanti-CD36 antibodies.

HeLa cells were labeled with PKH26-GL, followed by irradiation using a60UVB lamp [Derma Control Inc.], calibrated to provide 240 mj cm⁻² in 2minutes, sufficient for the induction of apoptosis. After 6-8 hoursimmature DCs dyed with PKH67-GL and pre-treated with 50 μg /ml ofvarious monoclonal antibodies for 30 minutes were added to the wellscontaining apoptotic HeLa cells. 45-60 minutes later, cells wereanalyzed by FACS® for double positive cells. Phagocytic uptake isreported as a percentage of untreated cells. Maximal phagocytosis rangedfrom 44-52%. Results from three experiments were averaged and meansplotted+SD. Similar results [data not shown] were obtained whenapoptotic monocytes were used [A]. Immature DCs were incubated with redfluorescent latex beads at 37° C. [squares], 4° C. [diamonds], or at 37°C. in the presence of 50 μg/ml anti-α_(v)β₅ [circles], anti-α_(v)[triangles]. The results shown indicate percent phagocytosis overbackground [B].

FIGS. 21A, 21B, 21C, 21D and 21E: DCs phagocytose apoptotic cells andnecrotic cells. 293 cells were labeled red with the PKH26 GL fluorescentcell linker and induced to undergo apoptosis via UVB irradiation, ornecrosis via repeated freezing and thawing. Immature DCs were dyed greenwith PKH 67-GL and then co-cultured with the apoptotic and necroticcells for three hours at 4° or 37° C. at a ratio of 1:1. Cells wereanalysed by FACScan where double positive cells indicate uptake of theapoptotic and necrotic cells. In another embodiment, the maturationfactor is selected from a group consisting of monocyte conditionedmedium; IFN-α and at least one other factor selected from the groupconsisting of IL-β, IL-6 and TNF-α; and necrotic cells by the DCs. Dotblots are gated on the FL1 high positive cells [DCs] thus excluding thedead 293 cells from the analysis. [A] Green dyed DCs and red dyedapoptotic [293 UV] and necrotic [293 FT] cells alone. [B] Uptake ofapoptotic and necrotic cells by immature DCs; [C] Uptake of apoptoticand necrotic cells by mature DCs. The results shown are representativeof ten different experiments. [D] Uptake of FITC labeled beads and CD83expression by immature DCs. [E] Uptake of FITC labeled beads by immatureDCs which were then induced to mature via MCM.

FIGS. 22A, 22B, 22C, 22D, 22E and 22F: DCs exhibit a mature phenotypefollowing exposure to necrotic but not apoptotic cells. [A] Apoptotic ornecrotic BLCL were co-cultured with DCs at ratios of 1:2 or 1:5. After48 hours, the DCs were stained extracellularly for CD83 andintracellularly for CD83 and DC-LAMP (27). [B,C] DCs were co-culturedwith apoptotic or necrotic cells at a ratio of 1:2 for 48 hours and thenstained extracellularly for CD83 and intracellularly for DC-LAMP. Valuesrepresent the averages of the geometric mean indices from three or moreexperiments. Arrowbars mark the standard deviation. Isotype matchedantibodies served as controls in all experiments [data not shown]. [D]Immature and mature DCs were stained with monoclonal antibodies to CD83,CD86, CD40 and HLA-DR. Apoptotic or necrotic cell lines [E] or theirrespective supernatants [F] were added to immature DCs. After 48 hoursthe DC were stained with monoclonal antibodies to CD83, CD86, CD40 andHLA-DR. Values represent the averages of the geometric mean indices fromthree or more experiments. Arrowbars mark the standard deviation.Isotype matched antibodies served as controls in all experiments [datanot shown].

FIGS. 23A and 23B: DCs gain heightened T cell stimulatory capacity afterexposure to necrotic but not apoptotic cells. Apoptotic or necrotic celllines were added at various ratios to day 5 or 6 immature DCs. After 48hours of co-culture, the DCs were assayed for their T cell stimulatorycapacity. [A] DCs were irradiated with 3000 rad using a cesiumirradiator [Ce 57] prior to addition to syngeneic T cells and 0.1 ng/mlSEA. After 3 days, 4 μci/ml ³H-Thymidine was added for 16 hours. [B]After coculture with apoptotic or necrotic cells, or their respectivesupernatants, DCs were fixed with 1% paraformaldehyde for 30 min at 4°C., washed extensively, and added in graded doses to 2×10⁵ allogeneic Tcells. After 4 days 4 uci/ml ³H-Thymidine was added for 16 hours.Immature and MCM matured DCs served as controls. Results arerepresentative of three or more experiments and the values shownrepresent the mean of triplicate wells.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to methods of delivering antigens to dendriticcells for processing and presentation to T lymphocytes. In particular,this invention relates to the use of apoptotic cells for the packagingand delivery of exogenous antigen to the MHC class I presentationpathway of dendritic cells for stimulation or tolerization of classI-restricted CD8⁺ T cells. The apoptotic cell-activated dendritic cellsand antigen-specific T cells produced according to the method of thisinvention may be used for various immunological interventions for theprevention and treatment of disease.

For the purpose of a more complete understanding of the invention, thefollowing definitions are described herein:

The term “apoptosis” means non-necrotic cell death, which can occurunder a variety of conditions including programmed cell death, exposureto ionizing and UV irradiation, activation of fas and other tumornecrosis factor receptor-related pathways, and drugs. Apoptosis ischaracterized by, inter alia, formation of “blebs” and vesicles at theplasma membrane, cell shrinkage, pyknosis, and increased endonucleaseactivity (20-21). Specific markers for apoptosis include, but are notlimited to, annexin V staining, propidium iodide staining, DNAladdering, staining with dUTP and terminal transferase [TUNEL].

The term “apoptotic cell” means any cell expressing a native or foreignantigen undergoing apoptosis due to any condition, including those whichususally are associated with causing neurosis. Thus, an apoptotic cellis identified based on its characteristics described above rather thanany method used leading to cell death. Similarly, the term “apoptoticcell fragments” means apoptotic cell material, bodies, blebs, vesicles,or particles other than whole apoptotic cells which contain antigen.

The term “necrosis” means a form of cell death resulting fromirreversible trauma to cells typically caused by osmotic shock orexposure to chemical poison, and is characterized by marked swelling ofthe mitochondria and cytoplasm, followed by cell destruction andautolysis (22).

The term “donor cell” means the apoptotic cell that delivers antigen todendritic cells for processing and presentation to T cells.

The phenomenon of “cross-priming” occurs when antigens from donor cellsare acquired by the host APCs such as dendritic cells and are processedand presented on MHC molecules at the surface of the APC for activationof antigen-specific T cells.

The phenomenon of “cross-tolerance” occurs when antigens from donorcells are acquired by host dendritic cells and are presented underconditions that are non-inflammatory (lack of inflammation or othermaturation stimuli) so as to cause antigen-specific unresponsiveness inT cells.

The term “antigen” means all, or parts thereof, of a protein or peptidecapable of causing an immune response in a vertebrate preferrably amammal. Such antigens are also reactive with antibodies from animalsimmunized with said protein. The potent accessory function of dendriticcells provides for an antigen presentation system for virtually anyantigenic epitope which T lymphocytes are capable of recognizing throughtheir specific receptors. Example 1, infra, demonstrates how dendriticcells can present viral antigen after acquisition from donor cellsundergoing apoptosis, and effectively activate CTL responses.

Sources of Dendritic Cells.

The dendritic cells used in this invention can be isolated as describedherein or by methods known to those skilled in the art. In a preferredembodiment, human dendritic cells are used from an appropriate tissuesource, preferably blood or bone marrow.

Mature dendritic cells can also be obtained by culturing proliferatingor non-proliferating dendritic cell precursors in a culture mediumcontaining factors which promote maturation of immature dendritic cellsto mature dendritic cells. Steinman et al. U.S. Pat. No. 5,851,756 andU.S. application Ser. 08/600,483 (WO 97/29182) report methods andcompositions for obtaining dendritic cells and are incorporated hereinby reference.

The dendritic cell precursors, from which the immature dendritic cellsfor use in this invention are derived, are present in blood as PMBCs.Although most easily obtainable from blood, the precursor cells may alsobe obtained from any tissue. in which they reside, including bone marrowand spleen tissue. When cultured in the presence of cytokines such as acombination of GM-CSF and IL-4 or IL-13 as described below, thenon-proliferating precursor cells give rise to immature dendritic cellsfor use in this invention.

Culture of Pluripotential PMBCs to Produce Immature Dendritic Cells.

Dendritic cell development can be divided into 4 stages: 1) aproliferating progenitor that can be either dendritic cell committed oruncommitted and capable of maturing to a nondendritic cell, 2) anon-proliferating precursor like the blood monocyte that does not showdendritic cell properties but is the starting population for manyclinical studies, 3) an immature dendritic cell which has properties andcommitment to become a dendritic cell, e.g. specialized antigen capturemechanisms including apoptotic cells for presentation, and MHC richcompartments, and 4) finally, the mature T cell stimulatory dendriticcell.

Cultures of immature dendritic cells, i.e. antigen-capturing phagocyticdendritic cells, may be obtained by culturing the non-proliferatingprecursor cells in the presence of cytokines which promote theirdifferentiation. A combination of GM-CSF and IL-4 at a concentration ofeach at between about 200 to about 2000 U/ml, more preferably betweenabout 500 and 1000 U/ml, and most preferably about 800 U/ml (GM-CSF) and1000 U/ml (IL-4) produces significant quantities of the immature, i.e.antigen-capturing phagocytic dendritic cells, dendritic cells. Othercytokines or methods known in the art which efficiently generateimmature dendritic cells may be used for purposes of this invention.Examples of other cytokines which promote differentiation of precursorcells into immature dendritic cells include, but are not limited to,IL-13. Maturation of dendritic cells requires the addition to the cellenvironment, preferably the culture medium,of a dendritic cellmaturation factor which may be selected from monocyte conditioned mediumand/or factors including TNF-α, IL-6, IFN-α, and IL-1-β. Alternatively,a mixture of necrotic cells or necrotic cell lysate may be added toinduce maturation.

Co-culture of Dendritic Cells with Apoptotic Cells

Apoptotic cells may be used to deliver antigen to either immature ormature dendritic cells, either freshly isolated or obtained from invitro culture. In a preferred embodiment, apoptotic cells comprising anantigen are co-cultured with immature dendritic cells for a timesufficient to allow the antigen to be internalized by the immaturedendritic cells. These immature dendritic cells are then caused tomature by the addition of a maturation factor to the culture medium. Thematured dendritic cells expressing processed antigen on their surfaceare then exposed to T cells for potent CTL induction.

In another embodiment, apoptotic cells may be used to deliver antigen tomature dendritic cells through surface receptors that enhanceinternalization of the apoptotic cells.

In another embodiment, apoptotic cells may be used to deliver antigen toimmature dendritic cells that are maintained as immature dendritic cellsas a means of efficiently modulating T cell tolerance or immunity insitu.

In a preferred embodiment, peripheral blood mononuclear cells [PBMCs]can be isolated from blood by sedimentation techniques. T cell-enriched[ER⁺] and T cell-depleted [ER⁻] populations can be prepared by resettingwith neuraminidase treated sheep red blood cells. Dendritic cells areprepared from the ER⁻ cells (Steinman et al. application Ser. No.08/600,483) as discussed above and are preferably cultured for 7 days to10 days in the presence of GM-CSF and IL-4. On about day 7 through 10,apoptotic cells can be co-cultured with the dendritic cells and thedendritic cells caused to mature over the next four days with theaddition of monocyte conditioned medium, a signal for maturation.

Besides monocyte conditioned medium, a combination of cytokines may beused to induce maturation of the immature dendritic cells. Examples ofcytokines which may be used alone or in combination with each otherinclude, but are not limited to, TNF-α, IL-1β, IL-6, IFN-α pathogens,autoimmune antigens and necrotic cells.

The apoptotic cell-activated dendritic cells made according to themethod described above are the most efficient for induction of CTLresponses. Delivery of antigen to mature dendritic cells, oralternatively, immature dendritic cells that are not caused to mature invitro, is also within the scope of this invention.

The apoptotic cells useful for practicing the method of this inventionshould efficiently trigger antigen internalization by dendritic cells,and once internalized, facilitate translocation of the antigen to theappropriate antigen processing compartment.

In a preferred embodiment, the apoptotic cells, or fragments, blebs orbodies thereof, are internalized by the dendritic cells and targeted toan MHC class I processing compartment for activation of classI-restricted CD8⁺ cytotoxic T cells.

In another embodiment, the apoptotic cells can be used to activate classII-restricted CD4⁺ T helper cells by targeting antigen via the exogenouspathway and charging MHC class II molecules. Apoptotic cells, blebs andbodies are acquired by dendritic cells by phagocytosis. When apopulation of CD4+ cells is co-cultured with apoptotic cell-primeddendritic cells, the CD4+ T cells are activated by dendritic cells thathave charged their MHC class II molecules with antigenic peptides. Theapoptotic cell-charged dendritic cells of this invention activateantigen-specific CD4+ T cells with high efficiency.

For purposes of this invention, any cell type which expresses antigenand is capable of undergoing apoptosis can potentially serve as a donorcell for antigen delivery to the potent dendritic cell system. Examplesof antigen that can be delivered to dendritic cells by donor cellsinclude, but are not limited to, viral, bacterial, protozoan, microbialand tumor antigens as well as self-antigens. Preferred antigens forpriming dendritic cells in vitro or in vivo are derived from influenzavirus, malaria, HIV, EBV human papilloma virus (including bothEPV-associated and EBV-unassociated lymphomas), CMV, renal cellcarcinoma antigens, and melanoma antigens. In addition, self antigensthat are targets of autoimmune responses can be delivered to dendriticcells e.g. insulin, histones, GAD.

For purposes of this invention the population of donor cells expressingantigen can be induced to undergo apoptosis in vitro or in vivo using avariety of methods known in the art including, but not limited to, viralinfection, irradiation with ultraviolet light, gamma radiation steroids,cytokines or by depriving donor cells of nutrient's in the cell culturemedium. Time course studies can establish incubation periods sufficientfor optimal induction of apoptosis in a population of donor cells. Forexample, monocytes infected with influenza virus begin to express earlymarkers for apoptotis by 6 hours after infection. Examples of specificmarkers for apoptosis include Annexin V, TUNEL+ cells, DNA laddering anduptake of propidium iodide.

Those skilled in the art will recognize that optimal timing forapoptosis will vary depending on the donor cells and the techniqueemployed for inducing apoptosis. Cell death can be assayed by a varietyof methods known in the art including, but not limited to, fluorescencestaining of early markers for apoptosis, and determination of percentapoptotic cells by standard cell sorting techniques.

In one embodiment, donor cells are induced to undergo apoptosis byirradiation with ultraviolet light. Depending on the cell type,typically exposure to UV light (60 mjules/cm²/sec) for 1 to 10 minutesinduces apoptosis. This technique can be applied to any cell type, andmay be most suitable for a wide range of therapeutic applications. Theapoptotic donor cells expressing an antigen of interest on their surfacecould then be used to prime dendritic cells in vitro or in vivo.

In another embodiment, donor cells are induced to undergo apoptosis byadministering a drug such as which induces apopotosis. This techniquecan also be applied to any cell type, and is also suitable for a widerange of therapeutic applications.

In another embodiment, donor cells are induced to undergo apoptosis byinfection with influenza virus. These apoptotic cells which expressviral antigens on their surface could then be used to prime dendriticcells in vitro or in vivo. The apoptotic cell-activated dendritic cellsmay then be used to activate potent influenza-specific T cells.

In another embodiment, tumor cells may be obtained and caused to undergoapoptosis. These apoptotic tumor cells, or tumor cell lines, could thenbe used to deliver tumor antigen to dendritic cells in vitro or in vivo.Once isolated, the tumor cells could be treated with collagenase orother enzymes which facilitate cell dissociation for culturing. Theapoptotic cell-activated dendritic cells may then be used as cancertherapeutic agents by activating the immune system to specificallytarget the tumor cells.

In another embodiment of the invention, the donor cells can betransfected, transduced or transformed to express foreign antigens priorto induction of apoptosis. In this manner dendritic cells may be loadedwith antigens not typically expressed on the donor cell. In addition,delivery of antigens via xenotransfer is also contemplated. Thesemethods can be accomplished using standard techniques known in the art.

A variety of possible antigens can be used in this invention including,but not limited to, bacterial, parasitic, fungal, viral pathogens,autoimmune antigens, and tumor antigens of cellular or viral origin.Preferred antigens include influenza virus, malaria, HIV, EBV, humanpapilloma virus, CMV, renal cell carcinoma antigens, and melanomaantigens breast cancer antigens, cancer antigens, and myeloma antigens.In addition, self antigens that are targets of autoimmune responses orother antigens for which it is desired to attenuate an immune responsecan be expressed on donor cells using any of the aforementioned methods.

Once donor cells expressing either native or foreign antigen have beeninduced to undergo apoptosis they can be contacted with an appropritenumber of dendritic cells in vitro or in vivo. The ratio of apoptoticcells to dendritic cells may be determined based on the methodsdisclosed in Example 1 or Example 6, infra.

For most antigens a ratio of only about 1-10 donor cells to 100 immaturedendritic cells is suitable for priming the dendritic cells. Highernumbers of apoptotic cells are preferred if mature dendritic cells areto be primed, preferably 100 to 1000 donor cells to mature dendriticcells.

The population of apoptotic cells should be exposed to the dendriticcells for a period of time sufficient for the dendritic cells tointernalize the apoptotic cell, or apoptotic cell fragments. Efficiencyof cross-priming or cross-tolerizing dendritic cells can be determinedby assaying T cell cytolytic activity in vitro or using dendritic cellsas targets of CTLs. Other methods known to those skilled in the art maybe used to detect the presence of antigen on the dendritic cell surfacefollowing their exposure to apoptotic donor cells. Moreover, thoseskilled in the art will recognize that the length of time necessary foran antigen presenting cell to phagocytose apoptotic cells, or cellfragments, may vary depending on the cell types and antigens used.

An important feature of the dendritic cells of this invention is thecapacity to efficiently present antigens on both MHC class I and classII molecules. Apoptotic donor cells, blebs, bodies or fragments thereof,are acquired by dendritic cells through the exogenous pathway byphagocytosis and as a result also efficiently charge MHC II molecules.CD4+ T cells may be activated by the dendritic cells presentingantigenic peptide which is complexed with MHC II using the methodaccording to this invention, since it is known in the art that dendriticcells are the most potent inducers of CD4+ helper T cell immunity. CD4+T cells can provide critical sources of help, both for generating activeCD8+ and other killer T cells during the acute response to antigen, andfor generating the memory that is required for long term resistance andvaccination. Thus, by using apoptotic cells to charge MHC class I and/orII products, efficient T cell modulation in situ can be achieved.

In a preferred embodiment both apoptotic and necrotic donor cells areused to prime dendritic cells. Preferably immature dendritic cells arecontacted with the donor cells and both class I and II MHC receptorsbecome optimally charged with antigen. In addition, the dendritic cellsare matured by the presence of the necrotic cells which also contributesto MHC class II loading.

A novel aspect of this invention is the use of apoptotic cells to primedendritic cells with antigen. An important advantage of the apoptoticcell system is that even poorly defined or undefined antigens can berouted to the appropriate antigen processing compartment of thedendritic cells to generate antigen-specific T cell responses. Moreover,dendritic cells can be charged with multiple antigens on multiple MHCsto yield poly or oligoclonal stimulation of T cells. Thus, since thestarting material can be obtained from virtually any tissue, the onlyrequirement for efficient targeting of potentially any antigen todendritic cells is the packaging of the antigen with apoptotic cells orapoptotic cell material.

Accordingly, the scope of this invention includes an embodiment wherebythe apoptotic cell delivery system is reconstituted in vitro usingapoptotic cell fragments, or material thereof, including, but not limitto, proteins, phospholipids, carbohydrates and/or glycolipids, whichenhance internalization and translocation of antigen to the appropriateantigen processing compartment in the dendritic cells. Thus, it iscontemplated that liposomes comprising at least antigen and anycombination of the aforementioned apoptotic cell fragments, or materialsthereof, may enhance delivery of antigen to dendritic cells. Liposomesenhanced with apoptotic cell fragments, or materials thereof inaccordance with this invention, can also be used for delivering antigenin vivo to dendritic cells.

As stated above, to prepare the liposomes it is desirable to includeligands for various receptors on the dendritic cells including variousintegrin receptors, CD36 and heat shock proteins. It is alsocontemplated that various mutant forms or fragments containing bindingdomains of such proteins may also be used. If DC are pulsed withliposomes in vitro, between 0.1-1000 ng of antigen is preferred forpriming between 1×10⁵ and 5 million DCs. Higher amounts of antigen inliposomes are used to deliver between about 10 ng to 10 μg perimmunization if DCs are to be pulsed in vivo.

In another embodiment, the apoptotic cell antigen delivery system can beused as a method for identifying and isolating poorly characterizedantigens. For example, a population of tumor cells can be induced toundergo apoptosis, the apoptotic tumor cells can then be co-culturedwith dendritic cells for a time sufficient to allow relevant immunogenicpeptides to be processed and presented on the dendritic cell surface.The immunogenic peptides can then be eluted from the MHC and otherpresenting molecules of dendritic cell surface as disclosed in U.S. Pat.No. 5,851,756 and purified using standard protein purificationtechniques known in the art including, but not limited to, HPLC and massspectrophotemetry.

Activation of Dendritic Cells and CTLs

In yet another embodiment of this invention, the apoptotic cell-chargeddendritic cells or the antigen-specific T lymphocytes generated bymethods described herein may be used for either a prophylactic ortherapeutic purpose. For activating T cells in an individual betweenabout 2×10⁵ and 2×10⁹ more preferably between 1 million and 10 millionapoptotic cell-activated mature dendritic cells should be administeredto an individual. The dendritic cells should be administered in aphysiologically compatible carrier which is nontoxic to the cells andthe individual. Such a carrier may be the growth medium described above,or any suitable buffering medium such as phosphate buffered saline(PBS). The mature dendritic cells prepared according to this inventionare particularly potent at activating T cells. For example, using priormethods of dendritic cells the ratio of dendritic cells to T cellsnecessary for strong T cell activation is about 1 dendritic cell to 30 Tcells whereas the ratio according to this invention is about 1 T cell to1000 dendritic cells. Thus, fewer dendritic cells are required. Foractivating T cells in vitro the ratio of dendritic cells to T cells isbetween 1:10 and 1:1000. More preferably, between 1:30 and 1:150.Between approximately 10⁶ and 10⁹ or more activated T cells areadministered back to the individual to produce a response against theantigen.

Dendritic cells may be administered to an individual using standardmethods including intravenous, intraperitoneal, subcutaneously,intradermally or intramuscularly. The homing ability of the dendriticcells facilitates their ability to find T cells and cause theiractivation.

By adapting the system described herein, dendritic cells could also beused for generating large numbers of CD8⁺ CTL, for adoptive transfer toimmunosuppressed individuals who are unable to mount normal immuneresponses. Immunotherapy with CD8⁺ CTL has been shown to amplify theimmune response. Bone marrow transplant recipients given CMV specificCTL by adoptive transfer, do not develop disease or viremia (4). Thesenovel approaches for vaccine design and prophylaxis should be applicableto several situations where CD8⁺ CTLs are believed to play a therapeuticrole e.g. HIV infection (1-3), malaria (5) and malignancies such asmelanoma (8-9).

Examples of diseases that may be treated by the methods disclosed hereininclude, but are not limited to bacterial infections, protozoan, such asmalaria, listeriosis, microbial infections, viral infections such as HIVor influenza, cancers or malignancies such as melanoma, autoimmunediseases such as psoriasis and ankolysing spondylitis.

By adapting the system described herein, dendritic cells could also beused for inducing tolerance. Dendritic cells expressing fas-L forexample can kill fas expressing activated T cells (23). Thus, it may bepossible to deliver fas-L bearing dendritic cells that are pulsed withapoptotic cells or blebs or bodies containing autoantigens to deleteautoreactive T cells in vivo; or alternatively, one can target dendriticcells that are specialized to induce tolerance rather than immunity, asproposed for the subset of dendritic cells known as “lymphoid dendriticcells” (see Steinman et al., Immunol. Rev. 156: 25-37, 1997).

Methods for Assessing Cytotoxic T Cell Activity

Frequently, in clinical disease it is difficult to detect killer cellsbecause of inadequate presentation. This invention also provides methodsfor assessing the cytotoxic activity of T lymphocytes, and in particularthe ability of cytotoxic T lymphocytes to be induced by antigenpresenting dendritic cells. According to this method, a samplecomprising T lymphocytes to be assayed for cytotoxic activity isobtained. Preferably, the cells are obtained from an individual fromwhom it is desirable to assess their capacity to provoke a cytotoxic Tlymphocyte response. The T lymphocytes are then exposed to antigenpresenting dendritic cells which have been caused to present antigen.Preferably, the dendritic cells have been primed with apoptotic cellswhich express antigen. After an appropriate period of time, which may bedetermined by assessing the cytotoxic activity of a control populationof T lymphocytes which are known to be capable of being induced tobecome cytotoxic cells, the T lymphocytes to be assessed are tested forcytotoxic activity in a standard cytotoxic assay. Such assays mayinclude, but are not limited to, the chromium release assay describedherein.

The method of assessing cytotoxic T lymphocyte activity is particularlyuseful for evaluating an individual's capacity to generate a cytotoxicresponse against cells expressing tumor or viral antigens. Accordingly,this method may be useful for evaluating an individual's ability todefend against cancers, for example melanoma, or viruses. In addition,this method is useful to detect autoreactive killer cells and couldmonitor not only the presence of killer cells, but their response totherapy.

All books, articles, or patents referenced herein are incorporated byreference in their entirety.

EXAMPLE 1 Use of Apoptotic Cells to Deliver Antigen to Dendritic Cellsand Induce Class I-Restricted CTLs Materials & Methods

Generation of mononuclear subsets. Peripheral blood mononuclear cells[PBMCs] were isolated from blood by sedimentation in Ficoll-Hypaque[Pharmacia Biotech]. T cell-enriched [ER⁺] and T cell-depleted [ER⁻]populations were prepared by resetting with neuraminidase treated-sheepred blood cells, as previously described (37). T cells were purifiedfrom ER⁺ cells by removal of monocytes, NK cells and MHC class II⁺ cells(37). Monocytes were obtained from ER⁻ cells by plastic adherence.Dendritic cells were prepared from ER⁻ cells cultured for 7 days in thepresence of GM-CSF and IL-4, followed by 4 days in monocyte conditionedmedium (42,43).

Induction and detection of apoptosis. Monocytes were infected withinfluenza virus in serum free RPMI. Initial time course studiesestablished that after 5 hours, monocytes began to express early markersfor apoptosis and that by 10-12 hours, a majority of the cells had beeninduced to undergo apoptosis. Cell death or Apoptosis was assayed usingthe Early Apoptosis Detection Kit [Kayima Biomedical]. Briefly, cellsare stained with Annexin V-FITC [Ann-V] and Propidium Iodide [PI]. Earlyapoptosis is defined by Ann V⁺/PI⁻ staining as determined by FACScan®[Becton Dickinson]. 293 cells were triggered to undergo apoptosis usinga 60 UVB lamp [Derma Control, Inc.], calibrated to provide 2 mJ/cm²/secat a distance of 8 cm for 10 mins.

Co-culture of DCs with Apoptotic Cells. Monocytes from HLA-A2.1⁻individuals were infected with influenza virus. Live influenza virus[Spafas,Inc.] was added at a final concentration of 250 HAU/ml [MOI of0.5] and incubated for 1 hour at 37° C. (37). Heat-inactivated virus wasprepared by incubating virus for 30 minutes at 56° C. (39). Cells werewashed with serum-containing medium and added to 24 well plates atvarying cell doses. After 1 hour, contaminating non-adherent cells wereremoved and fresh medium was added. Following a 10 hour incubation at37° C., 3.3×10³ uninfected DCs and 1×10⁶ T cells were added to thewells.

Assay for Virus-Specific CTLs. After 7 days of culture, T cells wereassayed for cytolytic activity using conventional Na⁵¹CrO₄ release assay(37). The targets were either influenza-infected syngeneic monocytes orT2 cells [a TAP^(−/−), HLA-A2.1⁺, class II⁻ cell line] pulsed for 1 hourwith 1 uM of the immunodominant influenza matrix peptide, GILGFVFTL (59,60). Specific lysis was determined by subtracting the percentcytotoxicity of uninfected monocytes or unpulsed T2 cells. Additionally,syngeneic DCs were used as targets where noted. In such cases, directpercent cytotoxicity is reported.

Reagents. The Z-VAD-CHO [Kayima Biomedical] was added to monocytes for 1hour prior to infection and subsequently washed out. DCs were culturedfor 1 hour in Brefeldin A (BFA) [Sigma] or NH₄Cl [Sigma] and thenmaintained in these inhibitors for the course of the assay.

RESULTS

Influenza A virus establishes a nontoxic infection in human DCs (37,38,2016-4008). Once infected, these DCs are capable of elicitingvirus-specific recall CTL responses within 7 days. The CTLs generatedare class I-restricted and kill virus-infected monocytes and peptidepulsed target cells (37,39). Compared with DCs, influenza infectedmonocytes are poor stimulators of CTL responses (37) and undergoapoptosis (40,41). We have exploited these observations to investigatethe role of apoptosis in the generation of antigen which could beacquired by uninfected DCs.

DCs were prepared from peripheral blood precursors of HLA-A2.1⁺individuals (42,43). Uninfected DCs and syngeneic T cells wereco-cultured with influenza-infected syngeneic [FIG. 1a] or allogeneic[FIG. 1b] monocytes for 7 days. Influenza-specific CTLs were generatedin these co-cultures, suggesting that the DCs acquired antigen from themonocytes. The DCs and not the infected monocytes functioned as the APC,since the latter failed to stimulate CTLs in the absence of DCs, even ata stimulator: responder ratio of 1:2 [FIG. 1a, dashed lines]. The CTLresponses generated in these co-cultures were as potent as those inducedby influenza-infected DCs. In some experiments, as few as 5×10³ infectedmonocytes were sufficient to charge uninfected DCs for the induction ofrobust CTLs [FIG. 1b]. Co-culturing influenza-infected HeLa cells withuninfected DCs also induced potent influenza-specific CTLs [data notshown]. The CTLs generated were capable of lysing influenza-infectedsyngeneic macrophage targets [FIG. 1a] as well as matrix peptide-pulsedHLA-A2.1⁺ T2 cells [FIG. 1b].

To confirm that antigen is transferred to, and expressed directly by, DCMHC class I products, we co-cultured uninfected DCs andinfluenza-infected monocytes, and used the DCs as targets for CTLs [FIG.1c]. DCs co-cultured with infected monocytes were as efficient asvirus-infected DCs at presenting antigen. These results suggest thatinfluenza antigen from infected monocytes gained access to MHC class Iof the DC, i.e. antigen from HLA mismatched monocytes were‘cross-priming’ T cells via the DC.

Given the nature of the antigen, it was important to exclude live virusas the agent responsible for the transfer of antigen to DCs. Based onprevious studies (14,20), influenza-infected monocytes produce only lowlevels of infectious virus before they undergo apoptosis. Infection of5×10⁴ monocytes with an MOI of 2.0 would be expected to yield up to1×10³ infectious virions after a 24 hour culture period. To preventinfection of DCs by monocyte produced virus, all experiments were donein human serum. The presence of blocking anti-hemagglutinin antibodiesprevents influenza virions from binding to cell surface glycoconjugates.Indeed, the addition of influenza virus [highest dose, 10⁴ infectiousvirions] directly into cultures containing uninfected DCs and T cellsdid not generate a CTL response [data not shown]. To further exclude thepossibility that monocytes were releasing virus during the 7 dayco-culture, we tested for the presence of free virions using a standardhemagglutination assay with chicken RBC (45). Less than 1 HAU/ml[hemagglutination units per milliliter] was detected in the medium at 12hr, 24 hr and 7 days of co-culture.

We also exclude the possibility that the CTL responses were due to freepeptide being released by the dying monocytes, thereby charging class Imolecules on DCs. Media from wells containing the infected monocyteswere collected after an overnight culture and transferred to fresh wellscontaining T cells and uninfected DCs. Virus-specific CTLs weregenerated after a 7 day culture period [FIG. 2a, 10 hr transfer].However, this CTL activity was abrogated if the medium was passedthrough a filter [0.45 um pore size] prior to being added to T cell-DCcultures [FIG. 2a, filter], suggesting that the antigenic material wasneither live virus nor free peptide, as both would have passed throughthe filter. This was confirmed by sedimentation experiments [FIG. 2b].Media from wells containing the infected monocytes were removed and spunat 250×g. The antigenic material could be localized in the pellet [FIG.2b, down arrow], but not in the supernatant fraction [FIG. 2b, uparrow]. Notably, the pelleted material fully accounted for the CTLactivity generated in direct transfer [FIG. 2b, stars]. Hemagglutinationactivity could not be detected in the pelleted material, confirming thatinfluenza virions were not trapped in this fraction. Furthermore, whenthe medium from infected monocyte cultures was pulsed onto T2 cells,they could not be targeted by influenza-specific CTLs [data not shown].Collectively, these results are most consistent with the source ofantigen being fragmented or intact apoptotic cells.

The role of apoptosis in the transfer of antigen to the uninfected DCwas therefore assessed. We first show that influenza-infected monocytesundergo apoptosis as detected by annexin V binding, an early marker forprogrammed cell death [FIG. 3a]. In contrast, heat-inactivated influenzavirus, which reliably reduces viral replication, failed to induceapoptosis in monocytes [FIG. 3a]. However, DCs that are infected withheat-inactivated virus remain capable of stimulating potentinfluenza-specific CTL responses. We employed this replication deficientvirus to probe the requirement for apoptosis in cross-priming. Whenmedia from wells containing the heat-inactivated influenza-infectedmonocytes were collected after an overnight culture and transferred tofresh wells containing T cells and uninfected DCs, no influenza-specificCTLs were generated [FIG. 3b, 10 hr transfer, HI virus]. In contrast,DCs exposed to medium derived from live virus-infected monocytes, whichcontained apoptotic cells, did elicit a CTL response [FIG. 3b, 10 hrtransfer, live virus]. However, if the heat-inactivatedinfluenza-infected monocytes were allowed to undergo apoptosisspontaneously, as occurs during 7 days of culture (46), antigenicmaterial was generated and virus-specific CTLs were induced [FIG. 3b,co-culture, HI virus]. This data also argues against the possibilitythat live virus within the apoptotic cell is responsible for thetransfer of antigenic material to the DCs.

To establish that apoptosis is the trigger for cross-priming, we firstused Z-VAD-CHO, irreversible peptide inhibitor of caspase activity[Kamiya Biomedical Company]. It was possible to block apoptosis ofinfluenza-infected monocytes [determined by TUNEL], without affectingthe expression of viral proteins, [evaluated by staining withanti-nucleoprotein antibodies, data not shown]. Influenza-infectedmonocytes were cultured overnight in the presence of varyingconcentrations of Z-VAD. The medium from these wells were then added tofresh wells containing T cells and uninfected DCs. After 7 days, theresponding T cells were measured for their influenza-specific cytolyticactivity. At concentrations of Z-VAD which inhibited apoptosis of theinfected monocytes, antigenic material was not transferred to theuninfected DCs and virus-specific CTLs were not generated [FIG. 3c].

We next compared apoptotic death to necrotic death for the generation ofantigenic material. Influenza-infected 293 cells were used for thisexperiment. Greater than 95% of the cells expressed influenza proteinsafter 10 hours, but unlike monocytes, apoptosis was not triggered.Apoptosis was induced in the 293 cells by exposure to 60 mJ/cm² UVBirradiation (47), and necrosis was achieved by incubation in a hypotonicsolution for 30 minutes at 37° C. Apoptotic or necrotic cells wereco-cultured with uninfected DCs and T cells for 7 days and responding Tcells were tested as described above. Only apoptotic but not necroticcells were capable of transferring antigen to the DCs, as determined bythe generation of a potent influenza-specific CTL response [FIG. 3d].Similar findings were made when apoptotic versus necroticinfluenza-infected monocytes were used as the source of antigen [datanot shown].

The cellular requirements for generating CTLs via this pathway were alsoinvestigated. Given that lysis of T2 cells, an HLA-A2.1⁺ cell line, wasdependent on the matrix peptide, which is specific for HLA-A2.1, it wasexpected that the effectors were MHC class I-restricted [FIG. 1b].Indeed, when CD4⁺ and CD8⁺ subpopulations were purified at the end ofthe 7 day culture period, CTL activity was detected only in the CD8⁺fraction [data not shown].

We next examined whether co-cultures of uninfected DCs,influenza-infected monocytes and purified CD8⁺ T cells could generateCTLs [FIG. 4a]. Purified CD8⁺ cells did not develop into potent CTLs in3 separate experiments even when exposed to high doses ofinfluenza-infected monocytes. The addition of CD4⁺ T cells, but nothuman IL-2, restored the ability to generate influenza-specific CTLs.Thus, CD8⁺ T cells require CD4⁺ T cell help during the induction phasein this exogenous pathway, but once generated they are the soleeffectors. Notably, DCs directly infected with influenza virus stimulaterobust CTL responses from purified CD8⁺ T cells, independent of CD4⁺ Tcell help or exogenous cytokines at stimulator: responder ratios as lowas 1:100 (37). The requirement for CD4⁺ T cells in this exogenouspathway distinguishes it from the endogenous class I pathway. Thisobservation is consistent with recent findings where CTL induction bycross-priming in vivo was shown to require CD4⁺ T cell help, by directaction on the APC (24).

DCs and macrophages were compared as potential mediators of thisexogenous pathway for class I MHC presentation. Uninfected HLA-A2.1+ DCsor macrophages, and syngeneic T cells were co-cultured with infectedHLA-A2.1− monocytes. After 7 days, the responding T cells were measuredfor their influenza-specific cytolytic activity. DCs, but notmacrophages, were capable of stimulating influenza-specific CTLs [FIG.4b]. In fact, as increasing doses of syngeneic uninfected macrophageswere introduced into co-cultures containing uninfected DCs, T cells andinfected allogeneic monocytes, the CTL activity was abrogated [FIG. 4b].Presumably, the macrophages act to sequester antigen from the DCs byefficiently engulfing the apoptotic cells.

Different pathways have been proposed for the presentation of exogenousantigen on MHC class I molecules. One possibility is that the antigensaccess the cytoplasm directly from the endosomal compartments and enterthe ‘classical’ class I processing pathway (26,29). Alternatively, classI molecules associated with invariant chain may enter the endosomes(49). Finally, peptides generated in the endosomes may be regurgitatedto the cell surface, charging class I molecules (34). We investigated ifintracellular processing was required for DCs to present antigens fromapoptotic cells. Uninfected DCs pulsed with influenza-infectedallogeneic monocytes were used as targets for influenza specific CTLs[Table I]. A 3-9 hr co-culture of uninfected DCs and infected apoptoticmonocytes was sufficient to charge DCs with antigen. The DCs werepreincubated with 10 mM ammonium chloride, which neutralizes theendocytic compartment, or 6 ug/ml Brefeldin A [BFA], an inhibitor ofvesicular transport (50). Both ammonium chloride and BFA completelyinhibited the DCs ability to present antigenic material on MHC class I.Lactacystin, an irreversible inhibitor of the 26S proteasome (51), onlypartially blocked antigen presentation by the uninfected DCs[unpublished data]. The data suggest that both classical andnon-classical class I pathways are utilized for the presentation ofexogenous antigen derived from apoptotic cells.

Since processing was required for presentation of monocyte derivedantigen, immunofluorescence and electron microscopy were used todocument that DCs phagocytosed apoptotic cells. By immunofluorescence,we observed phagocytosis of fragmented or-intact cell bodies in 10-20%of the DCs [FIG. 5a]. DCs were identified by the DC-restricted marker,p55, and apoptotic material was identified by intense DAPI staining ofpyknotic nuclei. This was confirmed by electron and immuno-electronmicroscopy [FIGS. 5b,c]. Within the cytoplasm of 20-25% of the DCs,identified by expression of CD83 [FIG. 5c, inset 1], we observedapoptotic cells with an apparently intact plasma membrane [inset 2]. Webelieve these images represent the method by which DCs acquire apoptoticcells. These results are consistent with a recent report where it wasreported that DCs associate with apoptotic cells via the vitronectinreceptor αvβ3, but fail to associate with opsonized particles andnecrotic cells (52).

Previous studies have shown that murine DCs have the capacity to presentsoluble antigens via an exogenous pathway, leading to the induction ofMHC class I-restricted CTLs (32, 53-55). In addition, a single reportreported that following intra-venous injection of allogeneic cells, ratinterdigitating DCs within lymphnodes were found to contain whole cellsand cell fragments (56). Using virus-infected monocytes, whichthemselves do not induce CTLs, we show that human DCs can acquirephysiologically relevant antigens by phagocytosing apoptotic cells andthen stimulating MHC class I-restricted CTLs.

EXAMPLE 2 Apoptotic Transfected 293 Cells Serve as Antigenic Materialfor ‘Cross-Priming’ of CD8⁺ T cells

The methods and materials unless otherwise specified were the same as inExample 1.

The following methods were used to carry out the experiments shown inFIG. 6. 293 cells, a human kidney epithelial cell line, were transfectedwith a construct encoding the matrix gene from Influenza A (filledtriangles). After 2 days in culture, 1×10⁴ transfected 293 cells wereadded to fresh wells and were UV-B irradiated in order to induceapoptotic death. DCs and T cells were then added to these wells andafter 7 days, responding T cells were assayed for influenza-specificcytolytic activity using matrix peptide pulsed T2 cells as targets.

Results

The results demonstrate that a transfected tumor cell line can serve asa donor apoptotic cell, allowing for the transfer of antigen to theuninfected dendritic cell and the effective induction ofantigen-specific CD8⁺ CTLs. Controls included: mock transfected cells(open triangles), infected and uninfected 293 cells (filled and opencircles, respectively), infected 293 cells without DCs (filled circles,dotted line), infected and uninfected DCs (filled and open diamonds,respectively).

EXAMPLE 3 CD8⁺ T-Cells and not CD4+ Cells are Responsible forInfuenza-specific Cytotoxicity

The following methods were used to carry out the experiments shown inFIG. 7. Generation of CD8⁺ CTLs requires CD4⁺ T-cell help. Purificationof CD8⁺ and CD4⁺ T cells after CTL induction, established that CD8⁺cells were responsible for the influenza-specific cytolytic activity.Influenza infected allogeneic monocytes were co-cultured with DCs andT-cells. After 7 days, subpopulations of T cells were purified andtested for cytolytic activity (37). Effector: Target ratio=15:1.

Results

Given that the lysis of T2 cells, an HLA-A2.1⁺ cell line, was dependenton the matrix peptide, which is specific for HLA-A2.1, it was expectedthat the effectors were MHC class I-restricted (see Example 1). Toconfirm this, highly purified CD4⁺ and CD8⁺ subpopulations were isolatedat the end of a 7 day culture period. As demonstrated in FIG. 7, CTLactivity was detected only in the CD8⁺ fraction.

EXAMPLE 4 Immature DCs Phagocytose Influenza Infected EL4 Cells andStimulate Influenza-specific CTLs

The following methods were used to carry out the experiments shown inFIG. 8. Immature DCs were pulsed vith various concentrations of UVirradiated influenza infected EL4 cells in the presence of monocyteconditioned media (a maturation signal for the DCs). After three days,the DCs were collected and cultured with 2×105 syngeneic T cells atresponder : stimulator ratios of 30:1 and 100:1 in a 96-well microtiterplate. After 7 days in culture, responding T cells were tested in astandard 51Cr release assay using T2 target cells pulsed with theimmunodominant epitope of influenza matrix protein. Controls includedthe use of UV irradiated uninfected EL4 cells as a source of apoptoticfood. Influenza infected DCs and EL4s served as positive and negativecontrols, respectively. Effector: target ratio was approximately 25:1.

Results

Immature DCs pulsed with apoptotic influenza infected EL4 cells in thecontext of monocyte conditioned media, were used to stimulate bulk Tcells. The immature DCs efficiently engulf the murine tumor cell lineand are able to cross-present influenza antigen derived from thoseapoptotic cells. Indeed, at a ratio of DCs:EL4s equal to 100:1, it wasstill possible to elicit potent T cells responses. This experiment alsodemonstrates the possibility of using xenotropic cells as a source ofantigen bearing apoptotic cells for the delivery of antigenic materialto the DCs.

EXAMPLE 5 Immature DCs but not Mature DCs and not MonocytesCross-present Apoptotic Material from Apoptotic Cells Allowing for theTargeting by Influenza-specific CTLs

The following methods were used to carry out the experiments shown inFIGS. 9A and 9B.

Immature DCs but not Mature DCs or Monocytes cross-present antigenicmaterial from apoptotic cells and become targets of antigen-specificCTLs. [FIG. 9A]. Various populations of HLA-A2.1+ professional antigenpresenting cells (APCs) were co-cultured with HLA-A2.1− influenzainfected monocytes. After 12 hrs, the APCs were loaded with ⁵¹Cr andused as targets for influenza-specific CTLs. Mature DCs were sorted byFACSort® (Becton Dickinson) by the DC-specific maturation marker CD83.Immature DCs were CD14− and sorted by FACSort® as a CD83− population.Mature monocytes were generated by culturing an adherent mononuclearcell fraction in a teflon beaker adherent for nine days. Immaturemonocytes are defined as cells cultured for two days post-plasticadherence. [FIG. 9B]. Controls included infected and uninfected APCs.HLA-A2.1−monocytes were also tested as targets to demonstrate theabsence of lysis when using a mis-matched target. Effector: TargetRatios=45:1 and 15:1. Specific Lysis=(% killing of APC cross-presentinginfluenza infected monocytes)—(% killing of APC cross-presentinguninfected monocytes). Background lysis ranged from 0-8%.

Results

To better define the antigen presenting cell capable ofcross-presentation of antigenic material derived from the engulfment ofapoptotic cells, an assay was established in which the APC is used as atarget for antigen-specific CTLs in a standard chromium release assay.Various populations of HLA-A2.1+ professional APCs were co-cultured withHLA-A2.1− influenza infected monocytes. After 12 hrs, the APCs wereloaded with 51Cr and used as targets for an influenza-reactive CTL line.Specific lysis indicates that the APC had cross-presented antigenicmaterial derived from the apoptotic cell, leading to the presence ofspecific peptide-MHC class I complexes on its surface.

While mature DCs were capable of serving as targets when infected withinfluenza, they were unable to engulf apoptotic cells presumably becausethe mechanisms of antigen uptake, phagocytosis in this case, had beendown regulated. Immature DCs co-cultured with apoptotic allogeneicinfluenza-infected monocytes were capable of serving as targets. Whencultured in the presence of conditioned media, a maturation signal forthe DC, these cells upregulate costimulator molecules which apparentlypermit the DCs to serve as more efficient targets. Notably, neither themature or the immature macrophages cross-presented antigenic materialderived from the apoptotic cells. Unlike the mature DC population (whichalso do not serve as effective targets in this assay), these APCs arecapable of phagocytosing apoptotic cells, however the engulfed materialis most likely degraded and not cross-presented on MHC class I. When thevarious HLA-A2.1+ APC populations were infected with influenza, allserved as effective targets demonstrating that the differences describedabove were not a function of MHC class I down-regulation or an inabilityfor presentation of antigen.

The HLA-A2.1−monocytes which were used as a source of apoptotic cellswere also tested as targets for the influenza-specific CTLs in order toconfirm that the observed lysis was not due to killing of the allogenicinfluenza-infected monocytes. This result suggests that in vivo, it isthe immature DC which is responsible for the uptake of apoptotic cells.After maturation and migration to a T cell rich area of a primary orsecondary lymph organ, the DCs could stimulate CD8+ class-I restrictedCTLs. Also indicated by this result is that the preferable method forimmunotherapy would be to first co-culture immature DCs with apoptoticcells (a source of exogenous antigenic material) along with a maturationsignal (CM, TNFα, IL1β, IFN-α, or some combination of theaforementioned) prior to injection.

EXAMPLE 6 Efficient Presentation of Phagocytosed Cellular Fragments onMHC Class II Products of Dendritic Cells Materials & Methods

Mice. Adult 6-8 week mice [BALB/C, C57BL/6, BALB/C×DBA2 F1,C57BL/6×DBA/2 F1] of both sexes were purchased from Taconic Farms [NY]and Japan SLC [Hamamatsu, Japan].

Cells. DCs were generated from bone marrow progenitors by culture inrGM-CSF as described (86). The cultures were set up in 24 well plates[Costar, Cambridge Mass.; Nunc, Naperville Ill.] and used at d6 when thewells were covered with aggregates of immature DCs. By d7-8, aggregatesrelease mature DCs, about 10⁵/well (86). Immature but not mature DCs arephagocytic for several particulates (79). B blasts were induced withlipopolysaccharide [E.coli 0111:B4, Sigma Chemical, St. Louis Mo., 10,μg/ml] for 3-4d, or with anti-μ [Jackson Immunoresearch Labs, West GrovePa.; F[ab]′₂ goat anti-mouse IgM] and IL-4 [Gibco-BRL] for 2d. B cellswere spleen cells that were passed over Sephadex G10 columns anddepleted of T cells with antibodies and complement [thy-l, TIB 99; CD4,L3T4; CD8, TIB 211; all from ATCC]. In some cases, necrosis was inducedin the B cells either by freeze-thawing 3-4 times, or with anti-B220antibody [monoclonal J11d] and rabbit complement. Human cells wereplastic adherent, monocytes from peripheral blood mononuclear cells andEBV-transformed B cell lines. Apoptosis was induced by infection withinfluenza (95), or by exposure to LPS [10 ng/ml] or UV light.

Antibodies. The Y-Ae hybridoma was generously provided by Dr. C.Janeway, Yale University, New Haven Conn. and grown serum-free. TheIgG2b antibody was purified on protein A sepharose, biotinylated, andused in the FACS along with PE-streptavidin to detect MHC classII—peptide complexes (75,76) with a mouse IgG2b anti-human TCR antibodyas the non-reactive control [PharMingen, San Diego Calif.]. The cellswere double labeled for FACS and for microscopy with FITC antibodies toCD8, CD11c, CD86, I-Ab [AF6-120.1], 1-Ad [39-10-8], and I-E[14-4-4S][PharMingen].

Capture of cell-derived peptides in vitro. Bone marrow DCs weregenerated in 24 well plates for 6 days in GM-CSF, gently washed toremove mature floating DCs, and then fed B blasts, human cells, or apeptide [residues 56-73 of I-Eα]. Before adding I-E⁺ cells or peptide,the DCs were immature, since much of the MHC class II was inintracellular lysosomal compartments and CD86 was not expressed at thesurface (84) After uptake of I-E, the DCs matured with high levels ofsurface MHC II and CD86.

Antigen presentation assays. The 20.6 T-T hybridoma recognizes the MHCII-peptide complex that is detected with Y-Ae mAb (81). Graded doses ofAPCs were added to 2×10⁴ hybridoma cells in flat bottomed microtestplates. At 24 h, supernatants were assayed for IL2 using a growth assayfor conA stimulated T blasts.

Capture of cell-derived peptides in vivo. 2×10⁶ H-2d BALB/C DCs wereinjected in PBS into the footpads of C57BL/6 [H-2b] or as a negativecontrol, CBA/J [H-2k] mice. After 2 days, the draining lymph nodes wereremoved, frozen in OCT embedding medium, sectioned, fixed in absoluteacetone 5 min, and stained successively with biotin-Y-Ae, avidinalkaline phosphatase conjugates, B220 for B cells, and peroxidaselabeled mouse anti-rat Ig. We then could visualize the position of blueY-Ae⁺ cells relative to B cells which are found in white pulp folliclesand scattered through the red pulp. To identify the Y-Ae⁺ cells, DCswere enriched from the nodes as described 25 and double labeled forbiotin Y-Ae and PE streptavidin [or nonreactive biotin IgG2b mAbascontrol] and different FITC conjugates to CD8, B220, CD11c, I-Ab, I-Ad.

Results

The development of many immune responses requires that antigenpresenting cells [APCs] present antigens that initially are expressed byother cells. This antigen transfer to APCs may often be essential, sincemost cell types lack the migratory and costimulatory properties requiredto initiate a T cell response. Examples of responses that can developfollowing antigen transfer, often termed cross-priming andcross-tolerance, include the rejection of malignant cells andtransplants, including xenografts (64-70), and the maintenance oftolerance to self tissues (71-73). However, underlying mechanisms havebeen difficult to identify directly. Prior experimental approaches haveutilized hematopoietic chimeras. In the chimera, the T cells that becomeprimed to transplanted or malignant tissue [or tolerized to self]recognize antigen presenting MHC products on bone marrow-derived cells.It is possible that antigen transfer simply involves the release ofpeptides from donor [tumor, transplant, self tissue] to recipient APCs.We have shown in the previous examples how human dendritic cells [DCs]present viral antigens from apoptotic infected cells to MHC class Irestricted, cytolytic T lymphocytes (74). In this example, we have usedan antibody to directly monitor the formation of specific MHC class IIpeptide complexes when DCs [which are bone marrow-derived] are exposedto other noninfected cells. In vitro, we find that antigen transferrequires phagocytosis, takes place with both xenogeneic and transformedcells. In vivo, where antigen transfer can be visualized with theantibody to MHC-peptide, DCs serve as a powerful donor and recipient ofcellular peptides. We propose that the steady state migration of DCs inafferent lymph, followed by processing by resident DCs in the T cellareas of lymphoid tissues, continuously tolerizes the T cell repertoireto self.

To demonstrate the capacity of DC to present peptides from other cells,we took advantage of the Y-Ae monoclonal antibody. Y-Ae sees an MHCclass II-peptide complex that is formed when one MHC II product, I-Ab,presents a peptide from residues 56-73 of another MHC II product, I-Eα(75,76). Actually, the repertoire of peptides presented on MHC productstypically include other MHC-derived peptides (77,78). We culturedC57BL/6 DCs [I-Eα⁻,I-Ab⁺] with BALB/C B cells [I-Eα⁺,I-Ab⁻] andmonitored the capacity of DCs to react with Y-Ae by cytofluorography.The DCs were from 6 day cultures of GM-CSF stimulated mouse bone marrow,since these are known to have phagocytic activity, in contrast to themature nonphagocytic DCs that predominate by day 7-8 of these cultures(79). The B cells were activated 3-4 with LPS or with anti-α plus IL-4to express high levels of MHC II (80). When returned to culture, 20-40%of the B blasts undergo apoptosis spontaneously, so that it isunnecessary to induce apoptosis in these I-E rich cells, in contrast tohuman monocytes (below) where the need for apoptosis is shown fortransfer of cell-associated antigens to DCs.

FIG. 10A illustrates data from >30 experiments. H-2b DCs were culturedfor 20 h without peptide, with 1 μM I-Eα peptide, or with 2×10⁶ B blastsfrom H-2d [I-Eα⁺] or H-2b [I-Eα⁻] mice. Y-Ae labeling developed whenpeptide was added. The labeling was highest on DCs that had matured toexpress high levels of the CD86 costimulator [black arrows denote matureDCs in each panel]. A Y-Ae signal that was comparable to that seen withpeptide also developed on most DCs when I-E⁺ [BALB/C] B blasts, but notI-E⁻ [C57BL/6] B blasts, were added [FIG. 10A]. No labeling occurredwith a nonreactive monoclonal, isotype-matched to antibody Y-Ae [FIG.10A, lower]. The Y-Ae⁺ cells had markers of DCs [CD11c⁺, 1-Ab⁺] but notdonor B cells [I-Ad⁺, CD86 low] [FIG. 10B]. No Y-Ae signal was detectedon B blasts alone, or if B blasts were added to H-2d DCs [not shown]. Inkinetic studies, the Y-Ae signal appeared after 5 hr of DC-B cellcoculture and reached a plateau at 15-25 hr [FIG. 10C]. The signal thenremained stable for 3 days, the longest we followed the cultures. Thedevelopment of a Y-Ae signal on DCs depended on the B cell dose, and 4day B blasts gave much stronger signals than unstimulated B cells [FIG.10D]. B blasts can have 10 fold higher levels of MHC II products thansmall B cells (80). LPS and anti-μ blasts gave similar signals [notshown], with a plateau at a ratio of 10 B blasts per DC [FIG. 10D].

The MHC II-peptide complexes that formed during antigen transfer from Bblasts to DCs could be recognized by T cells, as shown with a hybridomathat responds to the I-Ab/Eα peptide complex by secreting IL-2 (81).Following a 20 hour exposure to I-Eα peptide or B blasts, the DCs weresorted as CD11c/CD86⁺ cells, fixed in formaldehyde to block furtherprocessing, added in graded doses to the T-T hybridoma for 24 hrs, andthen the medium collected for testing in an IL-2 bioassay [3H-TdRuptake, FIG. 10E]. DCs obtained from DC-B blast co-cultures were potentAPCs for T cells, almost as potent as DCs pulsed with I-Eα peptide andDCs expressing endogenous I-Ab complexes [I-Ab×I-E⁺ F1 mice].

To rule out the possibility that B blasts were simply releasing peptideto the DCs, we showed that Y-Ae labeling did not develop if B blastswere separated from DCs by a Transwell filter [0.45μ pores], or if Bblasts were added to nonphagocytic DCs that had undergone maturation inthe bone marrow culture [FIG. 11A]. To prove that cellular processingwas involved, we tested if NH₄Cl, which neutralizes the acidity of theendocytic system, blocked Y-Ae epitope formation. 5 mM NH₄Cl partially,and 20 mM totally blocked Y-Ae development [FIG. 11B]; the inhibitionwas reversible [see below].

Since the immune response to xenografts also involves processing ofdonor cells by host APCs (70), we tested human cells as the donor ofpeptides to DCs. This was feasible with the Y-Ae monoclonal, since humanHLA-DRA chain [residues] has the identical sequence to mouse I-Eα 56-73(82-83). The Y-Ae epitope developed when mouse DCs were cultured witheither EBV-transformed, human B lymphoblastoid cell lines [FIG. 11D] orwith human monocytes [FIG. 11C]. For monocytes, we showed that apoptoticcells [induced by influenza infection or exposure to UVB light] wereprocessed more effectively than necrotic, freeze-thawed cells and thatliving monocytes were not processed [FIG. 11C]. Comparable experimentson the need for apoptosis were difficult with mouse B blasts, since manyof the B blasts died spontaneously in culture.

To demonstrate that phagocytosis preceded the formation of the Y-Aeepitope, B blasts were fed to DCs in 20 mM NH₄Cl. After 20 h, DCs wereFACS® separated from residual B blasts and recultured for 12 h withoutNH₄Cl. NH₄Cl completely blocked Y-Ae epitope development [FIG. 12A,top], even within saponin-permeabilized cells (not shown), but the Y-Aereappeared after removal of NH₄Cl [FIG. 12A, bottom]. The block imposedby NH₄Cl seemed primarily at the level of I-E processing, rather thanpeptide loading, since addition of I-Eα peptide for 20 h to NH₄Clcultures led to strong Y-Ae signals comparable to nonblocked cultures[FIG. 12B].

To determine whether this mechanism could function in vivo, we injecteddifferent I-A^(b−) I-E⁺ cell types [mouse splenocytes, B blasts and DCs;human monocytes] into the footpads of CS7BL/6 [I-A^(b+) I-E⁻] mice andthen examined draining lymph nodes 2 days later for Y-Ae⁺ cells. Thenegative controls were H-2k C3H recipients. only mature DCs, derivedfrom mouse bone marrow cultures (24), were found to be efficient donorsof I-E peptide to MHC II of host cells, with numerous Y-Ae⁺ cellsdeveloping in the DC-rich, T cell areas [FIG. 13A]. The finding waspursued by FACS analyses in which we examined cells that were enrichedfrom the lymph nodes as described (87) [FIG. 13B]. The Y-Ae epitope wasabundant, but again only in the situation where I-E⁺ DCs-from BALB/C orC3H/He mice were injected into I-Ab recipients [C57BL/6 but not C3H].The Y-Ae⁺ cells had the phenotype of recipient DCs, positive for I-Aband CD11c and negative for I-Ad, B220, and CD8 [FIG. 13B]. Most of thehost, I-Ab and CDll c⁺ DCs formed Y-Ae [FIG. 13B]. Since it is knownthat mature DCs are short-lived in culture (26) but in vivo can home tolymphoid organs prior to dying [reviewed in 27], we tested if live DCshad to be injected to observe antigen transfer. In fact, if most of theDCs were induced to apoptose by exposure to UV light just prior toinjection, only small amounts of Y-Ae were formed [FIG. 13B]. Thereforeby injecting mature living DCs, one presumably maximizes the interactionof donor and recipient DCs, the latter termed interdigitating cells, inthe T cell areas (90).

Many immune responses require that APCs present antigens from othercells (63-73). Most cells in the body do not migrate to the T cell areasof lymphoid tissues and lack costimulatory functions [like CD86], bothproperties being important for selecting and activating T cell-mediatedresponses. In contrast, DCs are a potent system for stimulating T cellimmunity and tolerance, having the required migratory and stimulatoryfunctions (91). This data, together with data on the presentation ofapoptotic, virus-infected cells on MHC class I (74) outline a newfunction for DCs, the presentation of antigens from other cells.

The consequences of antigen transfer to DCs, i.e., immunity[cross-priming] vs. nonresponsiveness [cross tolerance], may beinfluenced by stimuli in the DC environment. When DCs capture antigensfrom dying cells at a site of foreign antigen deposition, e.g., whenmicrobial agents infect and kill somatic cells, or when transplantsundergo rejection by what is termed the indirect pathway, the DCs may bealtered by an inflammatory stimulus to be immunogenic. On the otherhand, unresponsiveness or cross tolerance may develop when DCs captureantigens chronically from the normal, noninflammatory, apoptoticturnover of cells in vivo. Cross tolerance of self-reactive T cells isobserved, for example, when antigens from β cells of normal,noninflammed pancreatic islets are presented by APCs in draining lymphnodes (72,73).

Substantial numbers of DCs traffic constitutively in the afferent lymphto the T cell areas where they almost certainly die, because the numbersof DCs in lymphoid tissues are normally steady and DCs do not emergeinto the efferent lymph. Prior thinking has emphasized the potentialvalue of this migratory, short lived, patrolling DC population to pickup foreign antigens and then present these in the T cell areas. Ourresults suggest that migratory DCs are also being processed distributingtheir peptides to other DCs in lymphoid organs. In our experiments, itis unlikely that Y-Ae⁺ cells were first formed in the periphery since Bblasts, splenocytes, UV-treated DCs and human monocytes each did notlead to Y-Ae epitope formation in lymphoid organs. Instead, it appearednecessary for viable DCs to first migrate to the lymphoid organ, andthen the vast majority of host, I-Ab, lymph node DCs captured I-E⁺ donorDCs to form the Y-Ae MHC-peptide complexes. The recipient Y-Ae+ cells inthe T cell areas, sometimes termed “lymphoid DCs,” may be long-lived andare thought to have a regulatory function (90,92). In the steady state,i.e., in the absence of inflammatory signals, the continued processingof migratory DCs and any cellular antigens that they have acquired fromthe normal turnover of cells in tissues, could purge the T cellrepertoire of self reactivity (72,73), a function that is also carriedout on developing T cells by DCs in the thymus (93,94). As result, whenforeign inflammatory stimuli are to be presented to the immune system byDCs, there is less chance that autoreactivity to self antigens develops,since the same DC system has inactivated self reactive cells beforehand.

EXAMPLE 7 Immature Dendritic Cells Phagocytose Apoptotic Cells viaα_(v)β₅ and CD36, and Cross-present Antigens to CTLs Materials andMethods

Media. RPMI 1640 supplemented with 20 μg/ml of gentamicin [Gibco BRL],10 mM HEPES [Cellgro] and either 1% human plasma, 5% pooled human serum[c-six diagnostics] or 5% single donor human serum was used for DCpreparation, cell isolation and culture conditions (113,119).

Preparation of Cells. Peripheral blood mononuclear cells [PBMCs], DCs,macrophages and T cells were prepared as previously described(113,114,119). Briefly, peripheral blood was obtained from normal donorsin heparinized syringes and PBMCs were isolated by sedimentation overFicoll-Hypaque [Pharmacia Biotech]. T cell enriched and T cell depletedfractions were prepared by resetting with neuraminidase-treated sheepred blood cells (119). Immature dendritic cells [DCs] were prepared fromthe T cell depleted fraction by culturing cells in the presence ofgranulocyte and macrophage colony-stimulating factor [GM-CSF] andinterleukin 4 [IL-4] for 7 days. 1000 U/ml of GM-CSF [Immunex Corp.] and500-1000 U/ml of IL4 [Schering-Plough Corp.] were added to the cultureson days 0, 2 and 4. To generate mature DCs, the cultures weretransferred to fresh wells on day 7 and monocyte conditioned media [MCM]was added for an additional 3-4 days (113,114). At day 7, >95% of thecells were CD14⁻, CD83⁻, HLA-DR^(lo) DCs. On day 10-11, 80_(—)100% ofthe cells were of the mature CD14⁻, CD83⁺, HLA-DR^(hi) phenotype.FACSort® was used to generate highly pure populations of immature andmature DCs, based on their CD83⁻ and CD83⁺ phenotype, respectively.Macrophages were isolated from T cell depleted fractions by plasticadherence for one hour. After 24 hrs., cells were removed from theplates and placed in Teflon beakers for 3-9 days. T cells were furtherpurified from the T cell enriched fraction by removing contaminatingmonocytes, NK cells and B cells (119).

Antibodies. Antibodies to the following proteins were used: CD8-PE,CD14-PE, HLA-DR-PE, HLA-DR-biotin [Becton Dickinson], IgG2b [clone6603001, Coulter], CD8 [CRL 8014, ATCC], CD83 [clone HB15a, Coulter],MHC I [W6/32, ATCC clone HB95], CD36 [clone FA6, obtained from the Vthinternational workshop on leukocyte differentiation antigens], α_(v)[clone CLB-706, Chemicon International Inc.; clone 69.6.5, Coulter], β₁[clone 6S6, Chemicon International Inc.], β₃ [clone SZ21, Coulter; cloneRUU-PL 7F12, Becton Dickinson], β₅ [clone B5-IVF2, Upstatebiotechnology], α_(v)β₃ [clone 23C6, Pharmingen], α_(v)β₅ [clone P1F6,Chemicon International Inc.], CD71 [Dako], Mannose receptor [clone3.2PB1, a gift from A. Lanzavecchia], Nucleoprotein [ATCC clone HB85].

Induction of apoptotic death. Monocytes were infected with influenzavirus in serum free RPMI. These cells undergo viral induced apoptoticdeath within 6-8 hours. Cell death was confirmed using the EarlyApoptosis Detection Kit [Kayima Biomedical] (102). As previouslydescribed, cells are stained with Annexin V-FITC [Ann V] and PropidiumIodide [PI]. Early apoptosis is defined by Ann V⁺/PI⁻ staining asdetermined by FACScan® [Becton Dickinson]. To ensure that we werestudying the uptake of early apoptotic cells, the kinetics of death werecarefully worked out. 5-8 hours post-infection, monocytes firstexternalize PS on the outer leaflet of their cell membrane, as detectedwith Ann V. By 8-10 hours, these cells were TUNEL positive. It was notuntil 24-36 hours that the majority of the monocyte population includedtrypan blue into the cytoplasm, an indicator of secondary necrosis[unpublished data, (120,121)]. HeLa cells were triggered to undergoapoptosis using a 60 UVB lamp [Derma Control Inc.], calibrated toprovide 2 mJ/cm²/sec. The kinetics of cell death in these cells has beenpreviously defined (99).

Phagocytosis of apoptotic cells. Monocytes or HeLa cells were dyed redusing PKH26-GL [Sigma Biosciences], and induced to undergo apoptosis byinfluenza infection or UVB irradiation, respectively. After 6-8 hours,allowing time for the cells to undergo apoptosis, they were co-culturedwith phagocytic cells that were dyed green using PKH67-GL [SigmaBiosciences], at a ratio of 1:1. Macrophages were used 3-6 days afterisolation from peripheral blood; immature DCs were used on day 6-7 ofculture; and mature DCs were used on day 10-11. Where direct comparisonof cells was needed, cells were prepared from the same donor ondifferent days. In blocking experiments, the immature DCs werepre-incubated in the presence of 50 μg/ml of various monoclonalantibodies for 30 minutes prior to the establishment of co-cultures.After 45-120 minutes, FACScan® analysis was performed and doublepositive cells were enumerated.

Phagocytosis of latex beads. Immature DCs were preincubated at 37° C.with monoclonal antibodies specific for α_(v) and α_(v)β₅. 10⁶ cellswere then cultured with 5×10⁷ red fluorescent microspheres [diameter 1μ,2.5% solids, carboxylate modified latex; Sigma] for varying periods oftime. Alternatively, the cells were maintained 4° C. At the end of theassay, cells were separated from unengulfed beads by density gradientcentrifugation and analyzed by FACScan® analysis (122).

Immunofluorescence. Cells were adhered to Alcian Blue [Sigma] treatedcover slips and fixed in 100% acetone. Cells were stained withanti-influenza nucleoprotein antibody [HB85, ATCC] and Texas redconjugated goat anti-mouse IgG [Jackson ImmunoResearch]. This wasfollowed by staining with biotinylated anti-HLA-DR [Becton Dickinson]followed by FITC conjugated streptavidin [Jackson ImmunoResearch]. Cellswere visualized using an Zeiss Axioplan2 microscope.

Assay for cross-priming of apoptotic cells. Various APC populations wereprepared as described above from HLA-A2.1⁺ donors. Mature DCs werefurther purified by labeling with the DC-restricted marker CD83,followed by cell sorting on the FACSort® [Becton Dickinson]. ImmatureDCs were CD14⁻ and sorted by FACSort® as a CD83⁻ population. Maturemacrophages were generated by culturing an adherent mononuclear cellfraction in a Teflon beaker for 9 days. These APC populations wereco-cultured with HLA-A2.1⁻ influenza-infected monocytes. After 12 hr.,the APCs were loaded with Na⁵¹CrO₄, and used as targets forinfluenza-specific CTLs in a standard chromium release assay (102,119).Specific lysis indicates that the APC had cross-presented antigenicmaterial derived from the apoptotic cell, leading to the formation ofspecific peptide-MHC class I complexes on its surface. Specific Lysis=[%killing of APC cross-presenting influenza infected monocytes]—[% killingof APC cross-presenting uninfected monocytes]. Background lysis rangedfrom 0-8%. Controls included influenza infected and uninfected matureDCs, immature DCs and macrophages. The HLA-A2.1⁻ monocytes used as asource of apoptotic material were also tested as targets to demonstratethe absence of lysis when using a mis-matched target. For the controltargets, specific Lysis=[% killing of influenza infected APC]—[% killingof uninfected APC]. Background lysis ranged from 0-5%. Maximal influenzaspecific killing was determined using T2 cells [a TAP^(−/−), HLA-A2.1⁺,class II⁻ cell line] pulsed for 1 hr with 1 M of the immunodominantinfluenza matrix peptide, GILGFVFTL as targets (123). Responses variedfrom 25-60% as a function of the individual's prior exposure toinfluenza.

RT-PCR. RNA was purified from highly purified sorted cell populations ofimmature and mature DCs as described above. Messenger RNA for β₃, β₅ andCD36 were identified using an one step RT-PCR reaction [Titan kit,Boehringer Mannheim]. The forward primer 5′-TGAGAAGTGCCCCTGCCC was usedfor both β₃ and β₅. The reverse primers 5′-GTTGGCTGTGTCCCATTTTGCT and5′-TTGTAGGATTTGTGAACTTG were used for β₃ and β₅ to obtain 438 bp and 509bp products, respectively [primer sequences were generously provided byS. Silletti]. The forward primer 5′-GGGAATTCATATGAAATCATAAAAGCAACAAACATand the reverse primer 5′CGGAATTCTACATTTCACTTCCTCATTTTCTG for CD36yielded a product of 392 bp (124). The RT reaction was carried out for30 minutes at 56° C. followed by 30 cycles of amplification. After 30cycles of PCR the samples were visualized on an agarose gel.

Results

Immature DCs efficiently phagocytose apoptotic cells. Based on previousobservations that immature DCs are the cells responsible for capturingantigen (106), we predicted that apoptotic cells would be engulfed bestby immature DCs. To test this hypothesis, we established a phagocytosisassay which allowed us to visually detect the uptake of apoptotic cells,and compare the phagocytic capacity of immature DCs, mature DCs andmacrophages. Briefly, immature DCs were prepared by culturing a T-celldepleted fraction from peripheral blood in the presence of IL-4 andGM-CSF. Mature DCs were generated with the addition of monocyteconditioned medium [MCM] and these cells expressed the cell surface DCrestricted maturation marker CD83 (113,114,125). Macrophages wereprepared by culturing a plastic adherent cell population in Teflonbeakers for 3-9 days. As a source of apoptotic cells we used influenzainfected monocytes (102); virus infection induces apoptotic death inthese cells within 6-10 hours (102,120,121). Monocytes were first dyedred using PKH26-GL [Sigma Biosciences], and then infected with influenzavirus as previously described (119). After 6-8 hours, the various APCswere dyed green using the fluorescent cell linker compound, PKH67-GL[Sigma Biosciences], and co-cultured with the apoptotic cells at a ratioof 1:1. After 2 hours at 37° C., co-cultures of cells were analyzed byFACScan® analysis, allowing for quantification of phagocytic uptake asdouble positive cells. 80% of the macrophages, 50% of the immature DCs,and less than 10% of the mature DCs engulfed the apoptotic monocytesafter 2 hours of coculture [FIG. 14A]. The smear of double positivecells [PKH67 labeled APC that engulfed the PKH26 labeled apoptoticcells] indicates that both apoptotic bodies and whole apoptotic cellsserved as ‘food’ for the phagocytic cell [FIG. 14. panels iii., vi.,ix.]. Note that as the forward scatter of the APCs increased and thesetting of the FACS® shifted, the dying monocytes were excluded from theestablished region [FIG. 14. panels ii., v., viii.]. Maximal uptake byall APC populations was achieved within 2-4 hours and in part dependedupon the source of apoptotic cell used [FIG. 14B and data not shown].Given this kinetic data, we believe that macrophages and DCs engage andinternalize dying cells while they still display features of earlyapoptotic death. This data also demonstrates that it is the immature DCwhich preferentially acquires apoptotic material as compared to themature DC. The source of apoptotic cells was not critical, since weobtained similar results with UVB irradiated HeLa cells [FIG. 20 anddata not shown].

To confirm that this FACS® assay was measuring phagocytosis, we carriedout the assay at 4° C. and in the presence of inhibitors ofphagocytosis. Both low temperature [FIG. 15A], and cytochalasin D, aninhibitor of cytoskeletal function, blocked uptake [FIG. 15B].Phagocytosis by immature DCs also requires divalent cations as EDTA wasinhibitory [FIG. 15C]. To visually confirm the uptake recorded by FACS®,we prepared cytospins of the dyed co-cultures. The frequency of uptakecorrelated with that measured on FACS [data not shown]. We alsoperformed immunofluorescence on co-cultures of immature DCs, labeledwith anti-HLA-DR [DR], and apoptotic influenza infected monocytes,labeled with anti-influenza nucleoprotein [NP] [FIG. 16]. In the toppanel an apoptotic cell is seen just prior to being engulfed by a DC[large arrowhead]. Following phagocytosis, apoptotic cells were found inDR⁺ vesicles [small arrows], but not in the cytoplasm.

Only immature DCs cross-present antigen from the apoptotic cell on classI MHC. We next correlated the phagocytic capability of macrophages andDCs with their ability to cross-present antigenic material derived fromapoptotic cells. The cells were prepared from HLA-A2.1⁺ donors(113,114), co-cultured with HLA-A2.1⁻ influenza-infected monocytes for12 hr and then loaded with Na⁵¹CrO₄ for use as targets forinfluenza-specific CTLs (102,119). Specific lysis indicates that the APCcross-presented antigenic material derived from the apoptotic cell, byforming specific peptide-MHC class I complexes on its surface [FIG.17A]. As a direct comparison with the endogenous pathway for class I MHCpresentation, the same APC populations were infected with live influenzavirus and used as targets [FIG. 17B].

While mature DCs were efficient targets when infected with influenza,they were unable to cross-present antigens, presumably because they haddown regulated the ability to phagocytose the apoptotic monocytes. Theimmature DCs, however, did cross-present antigens from apoptotic cells.Furthermore, if the immature DCs were co-cultured with the apoptoticcells in the presence of MCM, a maturation stimulus, they were evenbetter targets. This is possibly due to the up regulation ofcostimulator and adhesion molecules 108,126), or the increased stabilityof peptide-MHC I complexes. Given that maximal uptake of apoptotic cellsby immature DCs occurs between 2-4 hours [FIG. 14B], we believe thatcross-presentation of apoptotic material reflects of the phagocytosisand processing of early apoptotic cells rather than secondary necroticcells [see Methods]. With respect to this issue, it is important torecognize that the influenza infected monocytes require 24 hours toundergo secondary necrosis [unpublished data, (120,121)].

Notably, macrophages which efficiently phagocytose apoptotic cells [FIG.14A], did not cross-present antigens to CTLs [FIG. 17B]. Presumably, theengulfed material is degraded, not cross-presented on MHC I. Thisprofound difference between the DC and macrophage populations issupported by our previous findings that macrophages do not cross-presentantigens from apoptotic cells during the induction phase of a classI-restricted antigen-specific T cell response. In fact, when put intoculture with DCs in a competition assay, they sequester the apoptoticmaterial and abrogate the CTL response (102).

Immature DCs can be distinguished from macrophages by intracellularexpression of CD83 and a unique profile of phagocytic receptors. Weinvestigated the possibility that immature DCs might phagocytoseapoptotic cells via pathways distinct from macrophages. To clearlydistinguish these cells we characterized them phenotypically. ImmatureDCs are distinguished by the absence of both CD14, a macrophagerestricted marker, and CD83, a maturation marker for DCs (125). We haveextended the use of CD83, finding that immature DCs can be distinguishedfrom both macrophages and mature DCs by their intracellular expressionof CD83 [FIG. 18]. Macrophages do not express CD83 intracellularly norextracellularly [FIG. 18], while mature DCs express CD83 bothintracellularly and extracellularly [FIG. 18].

These APC populations were examined for receptors involved inphagocytosing apoptotic material [Table 1]. These include: α_(v)β₃ andCD36 which act as co-receptors, for engulfment of apoptotic neutrophilsand lymphocytes by macrophages (127,128); and CD14 which has beenimplicated in the uptake of apoptotic cells by macrophages (129). Whilestudying the immature DC populations, we identified a discrepancy in theexpression of the α_(v) and β₃ integrin chains and investigated thepossibility that α_(v) was dimerizing with an alternate β chain. Usingantibodies which recognize combined epitopes of the α_(v)β₃ and theα_(v)β₅ heterodimers, we noted the selective expression of α_(v)β₅ onimmature DCs [FIG. 19A]. As is true for most receptors involved inantigen uptake (106,107), the expression of CD36, α_(v)β₅ and mannosereceptor on DCs is down regulated with maturation [FIG. 19B, Table 1].

TABLE I Receptor Profile of various Antigen Presenting Cells ImmatureDC^($) Mature DC^(#) Monocytes^(¥) Macrophages^(Δ) Macrophages^(Δ) Day 7Day 11 Day 0 Day 3 Day 9 CD8* 0-2 (3 ± 1)§ 0-3 (4 ± 1) 0-1 (4 ± 1) 0-5(3 ± 1) 1-2 (3 ± 1) CD14* 1-10 (4 ± 1) 0-6 (4 ± 1) 83-97 (149 ± 90)67-98 (61 ± 20) 49-90 (68 ± 48) CD83† 8-43 (8 ± 4) 73-99 (114 ± 15) 0-1(3 ± 1) 4-19 (4 ± 2) 2-14 (4 ± 2) MHC I† 99-100 (311 ± 49) 96-100 (280 ±69) 99-100 (99 ± 38) 97-99 (164 ± 24) 95-100 (116 ± 18) HLA-DR* 88-100(84 ± 35) 98-99 (188 ± 40) 94-99 (63 ± 9) 97-99 (173 ± 34) 5-89 (25 ±27) CD8† 3-9 (4 ± 1) 2-8 (3 ± 1) 0-2 (3 ± 1) 1-4 (3 ± 0) 1-5 (3 ± 1)IgG2b† 3-7 (3 ± 1) 2-7 (3 ± 1) 0-2 (3 ± 0) 1-4 (3 ± 1) 1-5 (3 ± 1) CD36†75-99 (75 ± 31) 20-50 (9 ± 2) 89-99 (62 ± 18) 64-64 (69 ± 45) 9-55 (15 ±16) α_(v) (CD51)† 45-76 (14 ± 3) 11-40 (9 ± 3) 2-12 (4 ± 1) 2-35 (6 ± 2)18-75 (10 ± 5) β₁ (CD29)† 99-100 (287 ± 57) 84-100 (112 ± 28) 95-100 (61± 33) 83-100 (72 ± 23) 68-99 (104 ± 29) β₃ (CD61)† 14-34 (7 ± 2) 10-24(6 ± 1) 76-91 (40 ± 16)^(∞) 15-85 (14 ± 9)^(∞) 19-92 (16 ± 9) β₅† 78-95(30 ± 8) 32-57 (11 ± 2) 6-8 (5 ± 1) 8-15 (7 ± 2) 5 8 (3 ± 1) α_(v)β₃(VnR)† 22-28 (7 ± 1) 14-30 (7 ± 2) 4-8 (4 ± 1) 7-27 (5 ± 2) 19-68 (10 ±4) α_(v)β₅† 81-90 (25 ± 5) 12-53 (9 ± 3) 1-14 (4 ± 1) 0-11 (3 ± 1) 6-15(4 ± 1) CD71† 75-99 (50 ± 29) 75-94 (61 ± 27) 3-7 (4 ± 0) 14-30 (7 ± 2)58-81 (30 ± 7) Mannose Receptor† 99-100 (272 ± 68) 38-80 (26 ± 11) 2-7(4 ± 0) 24-63 (13 ± 8) 48-95 (34 ± 20) *PE-conjugated. †Unconjugatedantibody. §Results are expressed as range of values, calculated aspercentage of positive cells as compared to an isotype matched controlantibody. Values in parentheses indicate the average of geometric meanfluorescence intensities of the various samples. ∞The β₃ expression inthe monocytes and day 3 macrophages does not match the α_(v)β₃expression due to contaminating platelets which stuck to the cellsduring isolation, as determined by staining with anti-CD41, data notshown. ^(¥)n = 4. ^(Δ)n = 5. ^(#)n = 6. ^($)n = 7.

To evaluate whether this down regulation could be observed on the levelof mRNA expression, we performed RT-PCR using primers specific for β₃,β₅ and CD36 [FIG. 19C]. Immature DCs [lane 1] showed amplified DNA ofthe appropriate size for β₃, β₅ and CD36. In contrast, in mature DCs[lane 2] no β₅, and much less CD36 sequences were seen, while β₃sequences were comparable to that in immature cells. These data, whilenot quantitative, are consistent with the levels of protein expressionobserved by FACS® and suggests that phagocytic receptor expression inDCs may be regulated at a transcriptional level as mRNA expression ofCD36 and β₅ is down regulated during maturation.

α_(v)β₅ and CD36 mediate phagocytosis of apoptotic cells in immatureDCs. To demonstrate a direct role for α_(v)β₅ in the recognition ofapoptotic cells by immature DCs, we performed the phagocytosis FACS®assay in the presence of antibodies specific for α_(v)β₅ [FIG. 20]. Inaddition to the blocking observed using the monoclonal to α_(v)β₅,blocking was also detected when using monoclonal antibodies to α_(v), β₅and CD36. Blocking was not observed when isotype matched monoclonalsspecific for β₁, β₃ or the transferrin receptor, CD71. Note, controlantibodies were chosen which recognized surface receptors present on theimmature DC [FIG. 20A, Table 1]. Monoclonal antibodies were tested indoses ranging from 10-80 μg/ml [data not shown]. Maximal inhibition ofphagocytosis of apoptotic cells was seen with mAbs specific for CD36,α_(v), and β₅ at 50 μg/ml. The inhibition of phagocytosis of apoptoticcells by DCs was specific. We were unable to block the uptake redfluorescent latex beads, a control particle, by DCs in the presence ofthese monoclonal antibodies [FIG. 20B]. By histogram analysis, DCsphagocytose 1-6 particles per cell. MAbs to α_(v)β₅ or α_(v) did notalter the profile of these histogram plots [data not shown].

While some inhibition of phagocytosis was observed when using α_(v)β₃this may in part be due to transdominance and/or the effect on the poolof free α_(v) (130). For example, anti-α_(v)β₃ antibodies suppress theintracellular signaling of the α₅β₁ integrin (131). Alternatively,α_(v)β₃ and α_(v)β₅ may be working cooperatively in the immature DCs. Wetherefore tested combinations of anti-α_(v)β₃ and anti-α_(v)β₅, but didnot observe an increase in the inhibition of phagocytosis. The lowreceptor density of α_(v)β₃ on DCs [average MFI of 7+/−2, Table 1] alsomakes it unlikely that this integrin heterodimer is involved in theengulfment of apoptotic cells by immature DCs.

Our data do not exclude a role for other receptors in the phagocytosisof apoptotic cells e.g. the putative phosphatidylserine receptor or thelectin receptor (100). In fact, other receptors are probably involved asblocking observed did not exceed 60%, even when combinations of allrelevant mAbs were tested [data not shown]. CD14 is unlikely to beinvolved in the DC's engulfment of apoptotic cells, as DCs do notexpress this receptor [Table 1]. In macrophages, phagocytosis ofapoptotic cells was inhibited by antibodies to α_(v), β₃, α_(v)β₃ andCD36 but not by antibodies to β₁, β₅ or α_(v)β₅ [data not shown]. Thiscorrelates with published data (101,128).

Cross-presentation of antigens to CTLs appears to have two criticalfeatures. It is mediated by DCs and apoptotic cells are the preferredsource of antigen (102). The requisite stage of DC development for theacquisition of apoptotic cells is the immature phase. In fact, immatureDCs, are 4-5 times more efficient than mature DCs in phagocytosis, afeature which also correlates with their ability to cross-presentantigen. This exogenous pathway for class I MHC loading is highlyeffective: relatively few apoptotic cells [ratio of 1 per 10 DCs] areneeded to charge the DCs as efficiently as the live replicating virus;exposure of 3-12 hours is sufficient for generating a peptide-MHCcomplex that is capable of activating CTLs; and it is relativelyindiscriminate, as the cellular source can be allogeneic cells orxenogeneic cells (102,132). We believe our earlier studies with matureDCs are explained by the fact that our cell populations wereasynchronous and that only by sorting these cells have the differencesbecome apparent. Based on the findings presented here, we suggest thatthe peripheral tissue DC, exemplified by the immature DC, has anadditional important role. It is responsible for phagocytosing cellswithin tissues which undergo apoptosis [e.g. secondary to viralinfection; during normal cell turnover] and migrating to the draininglymph nodes where appropriate T cells are engaged. This pathway may beemployed for stimulating or tolerizing CTLs and can account for the invivo observations of cross-priming of tumor and viral antigens (104,133)and cross-tolerance of self proteins (105,134) in their requirement fora bone-marrow derived APC.

A sharp distinction was also demonstrated between immature DCs andmacrophages in the handling of apoptotic material. While macrophages aremore efficient at phagocytosing apoptotic cells than immature DCs, theyfail to induce virus-specific CTLs (102). In fact, they cannot evengenerate effective levels of peptide/MHC I complexes. In a short-termassay, influenza-specific CTLs could not kill macrophages co-culturedwith apoptotic cells. Therefore, macrophages degrade rather thancross-present the ingested apoptotic cells.

Additional evidence exists that macrophages process apoptotic cellsdifferently from DCs, and prevent an immune response. Two groups havedemonstrated that phagocytosis of apoptotic cells suppresses asubsequent inflammatory response to LPS stimulation. The macrophage'scytokine profile is skewed toward the synthesis of IL-10, IL-13 andTGF-β, while the production of proinflammatory cytokines such as TNF-α,IL-1β, and IL-12 is down-modulated (136,137). Therefore, the resolutionof inflammation is dependant on at least two pathways for removal ofapoptotic cells: via macrophages which subvert and suppressproinflammatory responses; and via DCs, which stimulate T cell responsesthat clear pathogens responsible for the induction of the apoptoticdeath.

The α_(v)β₅ integrin receptor may be pivotal in the distinctive handlingof apoptotic cells by immature DCs versus. macrophages in that it'sexpression is restricted to the former. We suggest that the uniqueprofile of receptors expressed by immature DCs affects trafficking ofphagocytosed apoptotic cells, and consequently facilitatescross-presentation. We have previously shown that NH₄Cl inhibits the DCsability to process antigen derived from apoptotic cells, suggesting thatprocessing in an acidic vesicle [e.g. CIIVs or MIICs] is required(102,132). Indeed, class I MHC may interact with processed antigens insuch a compartment, as MHC I molecules have been described inassociation with invariant chain (138), and can recycle from the cellsurface to class II vesicles (139). Additionally, there may be as yetundescribed routes whereby antigens within vesicles can enter theclassical endogenous pathway as described recently for antigens derivedfrom the ER (140).

α_(v)β₅ and α_(v)β₃ have both been described to be important inangiogenesis, cell adhesion, migration and now in their ability tophagocytose apoptotic cells. α_(v)β₃ is critical in the phagocytosis ofapoptotic cells in macrophages, where it acts in a cooperative way withCD36 and thrombospondin [TSP], collectively forming a ‘molecular bridge’(142). Recently, it was reported that α_(v)β₅ but not α_(v)β₃ iscritical for the engulfment of rod outer segments [ROS] by CD36⁺ retinalpigment epithelial cells (143,144). This phagocytic system is alsoinhibited by anti-CD36 antibodies, suggesting that α_(v)β₅, likeα_(v)β₃, might cooperate with CD36. Taken together with ourobservations, TSP, or possibly other soluble factors, may serve tobridge CD36, α_(v)β₅ and the apoptotic cell.

Although similarities in function exist, α_(v)β₅ can be distinguishedfrom α_(v)β₃ in its use of various ligands [e.g. VEGF vs. bFGF]; by therequirements for activation; and by the intracellular signaling pathways[e.g. indirect activation of PKC] (50). Also significant is the factthat the cytoplasmic domains of the two β chains are the portions whichshow the most considerable diversity (51). Thus, it is possible that thedistinct use of the α_(v)β₅ vs. α_(v)β₃ integrin receptors might accountfor the specialized functions of DCs in the route by which apoptoticmaterial is trafficked and presented. In other words, differentialexpression of α_(v)β₅ may be responsible for the DCs ability tocross-present antigenic material derived from apoptotic cells, whereasmacrophages scavenge and degrade such material.

EXAMPLE 8 Inhibition of Integrin Inhibited Engulfment of Apoptotic Cells

We have shown in the previous Example that engulfment of apoptotic cellsby immature DCs is a receptor mediated phagocytic event, as it isinhibited by EDTA, Cytochalasin D and low temperature (see Example7)(147). Immature DCs express little α_(v)β₃, instead they employ analternate α_(v) heterodimer, the α_(v)β₅ integrin receptor, for theefficient internalization of apoptotic cells. As is the case in themacrophage receptor complex, α_(v)β₃/CD36, we believe that the α_(v)β₅integrin receptor works in concert with CD36. The expression of thisreceptor pair is restricted to the immature DC and upon maturation, CD36and α_(v)β₅ protein expression and their mRNA expression are both downregulated. This expression pattern fits what is known about DCbiology—the immature cell is involved in antigen capture, while themature stage is characterized by poor antigen acquisition, but insteadis efficient at activating T cells.

In Example 7, we demonstrated that the uptake of apoptotic humanmonocytes, HeLa cells, and a murine macrophage cells line (RAW) byimmature DCs are all inhibited by monoclonal antibodies specific forα_(v)β₅, α_(v), β₅ and CD36. While we do not have direct evidence forthe α_(v)β₅/CD36 interaction, we know that addition of blockingantibodies for both receptors to DCs/apoptotic cells co-cultures doesnot result in increased inhibition. This suggested to us that these tworeceptors might be working in concert. Maximal inhibition achieved inthe in vitro phagocytosis assay was never greater that 60%, suggestingthat additional receptors are involved. Candidates include, thecomplement receptors CR3 and CR4, scavenger receptor A, a putativelectin receptor (possibly DEC-205), and a still undefinedphosphatidylserine receptor (Reviewed in (149, 150)).

To better define the α_(v)β₅/CD36 receptor complex on immature DCs, wehave begun to search for a DC-restricted bridging molecule as it isunlikely that TSP-1 is the soluble factor involved in the α_(v)β₅/CD36receptor complex. Notably, neither the addition of antibodies specificfor TSP-1, nor the addition of purified TSP to DC/apoptotic cellco-cultures has an inhibitory effect (data not shown). In contrast, bothare known to inhibit engagement with CD36 on macrophages, thus blockingthe phagocytosis of apoptotic cells. Additionally, we have begun toinvestigate the role for IAP-1 in the activation of the α_(v)β₅integrin.

Based on the data described herein regarding the characteristics of aspecific molecular bridge between apoptotic cells and DCs we believethat Lactadherin, a major glycoprotein of the human milk fat globulemembrane, (151-153) has several properties which suggest it may be acomponent of such a molecular bridge. Lactadherin has many of theproperties required for acting as a molecular bridge between theimmature DC and the apoptotic cell as milk fat globules (MFGs) andapoptotic cells share much in common. The membrane surrounding the MFGhas the same orientation as the membrane of an apoptotic body/bleb (theouter leaflet of both is topologically identical to the plasmamembrane); the external leaflet of the membrane encapsulating thedroplet contains PS (likely due to a lack of flippase activity inherentto the MFG); and oligosaccharides comprised of fucose, galactose,N-acetylglucosamine and N-acetylgalactosamine (many of the samecarbohydrate motifs found on the surface of dying cells) (152-154). Themilk fat droplets are secreted from the lactating epithelial cells by aprocess of encapsulation in plasma membrane and is subsequently engulfedby the gut epithelium of the nursing child (155). Indeed, thecharacteristics which make Lactadherin successful at bridging the MFGwith the gut epithelium are similar to those which are likelyfacilitating uptake of the apoptotic cells by the DC. Supporting data: Arole for IAP-1.

Another proposed factor involved in the α_(v)β₅/CD36/Lactadherin complexis integrin associated protein-1 (IAP-1). IAP-1 is involved in theactivation of the α_(v)β₃/CD36/TSP-1 complex on macrophages. IAP-1 isexpressed on virtually all cells; it is present on immature DCs (datanot shown); and likely interacts with other α_(v) integrins as IAP-1 andβ₅ have been co-immunoprecipitated (195). To test the hypothesis thatIAP-1 is responsible for the activation of the α_(v)β₅/CD36 complex onimmature DCs, blocking studies with monoclonal antibodies and pertussistoxin (PT) were performed—we tested the effect of these factors on thephagocytosis of apoptotic cells by immature DCs (see Example 7 forexperimental details of phagocytosis assay).

Antibodies specific for IAP-1 (clone B6H12, Chemicon International Inc.)inhibited the phagocytosis of apoptotic cells, however the blockingobserved was significantly less than that observed when using anti-adantibodies (TABLE II). PT, the major virulence factor of Bordetellapertussis inhibits heterotrimeric G_(i)-proteins, also blocked thephagocytosis of apoptotic cells by immature DCs (TABLE II).

TABLE II Inhibitors of Phagocytosis* Added to Immature DC/ Apoptoticcell coculture Percent Inhibition (Std Dev.) anti-CD8 0 (n/a) anti-α_(v)46.5 (0.5) anti-IAP-1 27.8 (3.5) LB 40 μM^(†) 0 (n/a) LB 10 μM 0 (n/a)LA 40 μM 41.5 (1.5) LA 10 μM 14 (8) DMSO 0 (n/a) Herb 18 μM^(§) 66.5(2.5) Herb 9 μM 49 (2) Herb 1.8 μM 6.5 (2.5) PT 200 μg/ml^(∞) 37.5 (4.5)PT 50 μg/ml 19 (10) CC 12 μM^(≈) 4 (2) *Results are expressed asaverages of 2-4 experiments with values indicating percent inhibition ofphagocytosis of apoptotic cells as compared to respective control wells.Conrols were cocultures treated with anti-CD8, Lavendustin B (theinactive analog of Lavendustin A), or DMSO alone. Values in parenthesisindicate standard deviation. ^(†)Lavendustin B (LB) and Lavendustin A(LA). LA is a cell-permeable inhibitor of protein tyrosine kinaseactivity but has little effect on protein kinase A or protein kinase C.^(§)Herbamycin A is a potent cell-permeable protein tyrosine kinaseinhibitor and inhibits PDGF-induced phospholipase D activation (IC₅₀ = 8μg/ml) in a dose dependant manner. ^(∞)Pertussis Toxin (PT) is aninhibitor of the heterotrimeric G-protein G_(i). ^(≈)Chelethrinechloride (CC) is a cell-permeable inhibitor of protein kinase C (IC₅₀ =660 nM).

Taken together, our data suggests that α_(v)β₅/CD36/IAP-1/Lactadherinreceptor complex on immature DCs may be responsible for the efficientengulfment of apoptotic cells. Similar, but as yet unidentified factorscould also serve to enhance the uptake of apoptotic cells (or fragmentsthereof) in our antigen delivery system.

EXAMPLE 9 Consequences of Cell Death: Exposure to Necrotic but notApoptotic Cells Induces the Maturation of Immunostimulatory DendriticCells Materials & Methods

Preparation of DCs. DCs were prepared as previously described (171-173).In brief, DCs were generated from T cell depleted PBMCs by culturingcells for 5-6 days in the presence of 1,000 U/ml granulocyte andmacrophage colony-stimulating factor [GM-CSF, Immunex] and 1,000 U/mlIL-4 [Schering-Plough Corp. Union, N.J.]. RPMI 1640 supplemented with 20μg/ml gentamicin, 10 mM HEPES, [GIBCO] and 1% autologous plasma[heparinized] was used for cell culture. Cultures were supplemented withcytokines on days 2 and 4. On day 5-6 nonadherent immature DCs werecollected and transferred to new six well plates. Mature DCs weregenerated by the addition of 50% v/v monocyte conditioned medium [MCM]on the day of transfer and harvested on days 8-9.

Induction of Apoptosis and Necrosis. 293 cells were dyed red with PKH 26according to the manufacturer's protocol [Sigma Biosciences, St. Louis,USA]. UV-triggered apoptosis was induced using a 60 UVB lamp [DermaControl Inc]. Cells were incubated for 6 hours following irradiation toallow the cells to undergo apoptosis. Necrosis was achieved via repeatedfreezing and thawing. Immature DCs were dyed green with PKH 67 and thenco-cultured with the apoptotic and necrotic cells for 3 hours at 4° or37° C. Phagocytosis of apoptotic and necrotic cells by immature DCs wasdefined by the percentage of double positive cells by FACS-Analysis aspreviously described (176). UV-triggered apoptosis was also inducedusing a 60 mJ UVB lamp [Derma Control Inc.], calibrated to provide 2mJ/cm²/sec. Necrosis was achieved via repeated freeze [−80° C.] thawing[+37° C.]. Necrotic cells were trypan blue positive and demonstrated adistorted or fragmented morphology.

Cell lines. The following cell lines were used: Human cell linesconsisted of EBV transformed B-lymphocyte cell lines [B-LCL cells],melanoma cells [SK29 cells], and kidney adenocarcinoma cells [293 cells]and were cultured in RPMI supplemented with 10% FCS. Mouse cell lines[B16 melanoma cells, L-cells, RAW cells, 3T3 cells] were grown in DMEMsupplemented with 10% FCS.

Monoclonal antibodies. Monoclonal antibodies [mAbs] to the followingantigens were used. CD83 [Immunotech, Coulter, Marseille, France] andDC-LAMP [generously provided by Dr. S. Lebecque, Schering Plough,Dardilly, France] are markers expressed primarily by mature DCs. CD86,HLA-DR, CD40, CD25, CD8, CD14 were obtained from Becton-Dickinson[Mountainview, Calif.]. Isotype control mAbs included IgG1, IgG2a andIgG2b [Immunotech, Coulter, Marseille, France]. The secondary antibodywas PE-conjugated Fab′₂ goat anti-mouse IgG heavy and light chain[Jackson, ImmunoResearch, Laboratories, Inc., Baltimore, USA]. The DCcell populations were phenotyped with the panel of mAbs listed above andanalyzed on a FACScan [Becton Dickinson, Mountain View, Calif.]. Forintracellular staining cells were first fixed in 1% paraformaldehyde andthen permeabilized with 0.5% saponin before incubation with the primaryantibody.

Co-culture of apoptotic or necrotic cells with immature DCs. Apoptoticor necrotic cell lines were added at various ratios to day 5 or 6immature DCs. After 48 hours of coculture the DCs were assayed for theirT cell stimulatory capacity. For the superantigen dependent assay DCswere irradiated with 3000 rad prior to addition to syngeneic T cells inthe presence of 0.1 ng/ml SEA. After 3 days, 4 μci/ml ³H-thymidine wasadded for 16 hours. We observed background proliferation in 5 dayco-cultures, of immature DCs and allogeneic T cells in the MLR, probablybecause of some DC maturation as a result of DC-T cell interactions. Toavoid this, we fixed immature DCs after exposure to dying cells with 1%paraformaldehyde for 30 min on ice, washed extensively and added them ingraded doses to allogeneic T cells. After 4 days, 4 uci/ml ³H-thymidinewas added for 16 hours. Immature and MCM matured DCs served as controls.

Results

The immune system has to contend with two types of cell death and theirconsequences. Necrosis, characterized by cell fragmentation resultingfrom severe and sudden injury, leads to the release of toxicintracellular contents which may induce inflammation (156). Apoptosis,an energy dependent and synchronized event, is considerednon-inflammatory due to rapid scavenging by macrophages (156-158).Necrotic and apoptotic cells are also phagocytosed by dendritic cells,potent initiators of immunity, which induce T cell responses to antigensderived from these dying cells (159-161). Uptake is restricted to theimmature stage of development when DCs are well equipped to acquireantigen but express low levels of the requisite MHC and costimulatorymolecules needed for T cell stimulation (159). Upon receipt of amaturation signal, DCs downregulate antigen acquisition, express higherlevels of costimulatory and MHC molecules and become stablydifferentiated to activate resting T cells. Maturation can be triggeredby multiple stimuli including LPS, contact allergens, bacteria andviruses, cell products [monocyte conditioned medium-MCM, TNFα, IL-1β,PGE₂, IFN-alpha (162-168)] and signalling molecules [CD 40L (169-170)].Since it is the immature DC that captures antigen most efficiently, weinvestigated whether the uptake of dead or dying cells could initiateimmunity by inducing DC maturation.

To generate immature DCs, we cultured freshly isolated blood monocytesin GM-CSF and IL-4 for 6 days. These cells are characterized by lowlevels of HLA and co-stimulatory molecules [CD40, CD86 (159)] and the DCrestricted, maturation associated markers CD83 and DC-LAMP [lysosomalassociated membrane glycoprotein] (174). We first verified that immatureDCs phagocytosed necrotic cells comparably to apoptotic cells (175). AFACS assay was employed where green PKH26-GL labeled DCs are visuallyassessed for their ability to take up red PKH67-GL labeled apoptotic ornecrotic tumor cells [kidney adenocarcinoma, 293 cells] at a ratio of1:1 (176). Over 40% of immature DCs phagocytosed either apoptotic ornecrotic cells within 3 hours [FIGS. 21A,B,C]. Uptake was profoundlyreduced when dead cells were co-cultured with DCs at 4° C., indicatingthat cells or cell fragments were being bound specifically. As expected,mature DCs displayed poor phagocytic activity for either apoptotic ornecrotic cells [FIG. 21C]. As a control for the effects of phagocytosis,we added FITC-labeled latex beads to immature DCs which were thencultured for 48 hrs in the presence or absence of MCM, the latter toinduce maturation. Uptake of latex beads by itself did not inducematuration, as monitored by surface CD83 expression, and furthermore didnot inhibit maturation via MCM [FIGS. 21D,E (177)].

We next monitored the effects of dying cells on the maturation of DCs. Apanel of human cell lines was tested including a melanoma cell line[SK29 cells], kidney adenocarcinoma cells [293 cells] and EBVtransformed B-lymphocyte cell lines [B-LCL] [FIG. 22A,]. Again a 48 hrco-culture was carried out at different ratios of dying cells to DCs[1:5 to 1:2], after which the DCs were collected, stained for markersindicative of maturation, and analyzed on a FACScan®. Exposure tonecrotic cells, but not apoptotic cells, induced the expression of thematuration associated markers CD83 and DC-LAMP in a large percentage ofDCs [FIGS. 22A,B,C] and upregulated both co-stimulatory [CD86] and HLAmolecules [FIGS. 22D,E]. Furthermore, CD40 levels almost doubledfollowing exposure to necrotic cells [FIG. 22]. The induction ofmaturation was irreversible as DCs did not revert to an immaturephenotype in culture. Necrotic mouse cell lines, including L-cells, 3T3cells and B16 melanoma cells, also induced the maturation of DCs.Interestingly, when we added an excess of apoptotic cells to DCs atratios of 5:1 or 10:1 there was extensive DC death [data not shown]. Incontrast, this dose of cells was recently reported to induce maturationof a murine DC line (178), but the apoptotic cells were cultured for 16hrs prior to addition to DCs. It is possible that the apoptotic cellsunderwent secondary necrosis, and the latter induced maturation.

To define the nature of factors in necrotic cells that were responsiblefor inducing maturation, we collected supernatants from necrotic andapoptotic cell lines, filtered them through a 0.45 micron filter andadded these to immature DC cultures. Supernatants from necrotic but notapoptotic cells induced maturation [FIG. 22F]. Obvious candidates formaturation such as TNF-α and IL-1β were not detectable by ELISA in thesupernatants [not shown].

Mature DCs are potent stimulators of T cells, the most straightforwardassays being the induction of allogeneic T cell proliferation in themixed leukocyte reaction [MLR] and superantigen dependent T cellproliferation (179, 180). After a 48 hour exposure to dying cells ortheir respective supernatants, DCs were washed, and added in gradeddoses to 200,000 allogeneic or syngeneic T cells, in the latter casetogether with 0.1 ng/ml staphylococcus enterotoxin A [SEA]. Strongstimulation of allogeneic and superantigen stimulated syngeneic T cellswas observed with DCs that had been matured with necrotic cells, theirrespective supernatants or with MCM [FIGS. 23A,B]. In contrast, exposureto apoptotic cells did not induce MLR stimulatory activity orsuperantigen dependent syngeneic proliferation above the low backgroundin the absence of MCM [FIGS. 23A,B]. The ³H-thymidine uptake data wasconfirmed by analysis of T cell size on the FACS® scan. In response tomature DCs [matured with MCM or with exposure to necrotic cells], >30%of the CD3+ T cells in the MLR were blasts [high forward lightscattering], whereas immature DCs [no MCM, or exposure to apoptoticcells] induced <5% blast transformation [not shown]. Importantly, whenDCs were exposed to a mixture of apoptotic and necrotic cells, theyinduced similar increases in T cell stimulation as DCs cultured withnecrotic cells or their supernatants [data not shown]. Furthermore DCsexposed in parallel to apoptotic cells and MCM heightened T cellresponses to the same extent as DCs matured with MCM. These experimentsindicated that ingestion of apoptotic cells did not inhibit DCmaturation or function. This contrasts with recently published datashowing that phagocytosis of apoptotic cells induces immunosuppressiveand anti-inflammatory effects in macrophages [e.g. release of IL-10 andPGE₂ after LPS stimulation] and that apoptotic cells themselves can besources of factors [e.g. IL-10] that skew the immune response (182-183).

While a role for macrophages in the phagocytic clearance of bothapoptotic and necrotic cells is well known, there is little informationon how DCs respond to dying cells. We find striking differences in thehandling of dying, transformed cell lines depending on the mechanism ofcell death. Necrotic cells selectively induce maturation of DCs. Wewould predict that in vivo, uptake of necrotic tumor cells would lead tothe initiation of T cell responses to antigens processed by the DCs.These responses are likely to be CD4+ rather than CD8+, as antigensderived from necrotic cells do not induce CTLs, at least in vitro (160).Phagocytosis of apoptotic cells by DCs failed to induce maturation, theconsequences of which may be the induction of tolerance to self or tumorantigens (184-186). Phagocytosis of apoptotic cells, however, may leadto T cell immunity if followed by a maturation signal. We have shownthat DCs phagocytose apoptotic cells and present antigens [e.g. viraland tumor antigens] from these sources to both CD4+ and CD8+ T cells.See, Examples 1-5. Given that phagocytosis of apoptotic cells does notmature DCs, signals provided by necrotic cells in the enviroment, suchas cytokines [e.g. TNF-α, IL-1β, IFN-α released by virus infectedcells], inflammatory products [e.g. LPS, bacterial cell walls] and CD4+T cells [such as CD40-L/CD40 interactions (169, 170)] would be requiredto mature the DCs, thus allowing for the full activation of T cells. Infact in in vivo animal models of cross-priming and cross-tolerance,induction of CTLs requires CD4⁺ help [(184, 187-189) and data in vitronot shown].

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We claim:
 1. A method of delivering antigen to dendritic cells in vitro,said method comprising: contacting dendritic cells capable ofinternalizing antigens for presentation to immune cells with apoptoticcells comprising said antigen to be presented to immune cells whereinsaid contact is for a time sufficient to allow said antigen to beinternalized by the dendritic cells.
 2. The method according to claim 1wherein the dendritic cells are human.
 3. The method according to claim1 wherein the apoptotic cells are selected from the group consisting ofcells of a cell line, cells which have been transformed to express aforeign antigen, tumor cell line, xenogeneic cells, or tumor cells. 4.The method according to claim 3 wherein the apoptotic cells are selectedfrom the group consisting of monocytes, 293 cells, L cells, Hela cells,B cells and EL4 cells.
 5. The method according to claim 1 furthercomprising the step of inducing apoptosis of cells expressing saidantigen to produce the apoptotic cells.
 6. The method according to claim5 wherein apoptosis is induced by infection with influenza virus.
 7. Themethod according to claim 5 wherein apoptosis is induced by irradiationwith ultraviolet light, gamma radiation, steroids, serum deprivation,cytokines, or drugs which induce apoptosis.
 8. The method according toclaim 5 wherein said apoptotic cells are induced to become apoptotic invitro.
 9. The method according to claim 8 wherein apoptosis is inducedin vitro by depriving cells comprising said antigen of nutrients in thecell culture medium.
 10. The method according to claim 1 whereindendritic cells are exposed to a preparation of apoptotic cellfragments, blebs, or bodies containing antigen.
 11. The method accordingto claim 1 wherein said antigen is selected from a group consisting oftumor antigens, viral antigens, pathogens, microbial antigens, selfantigens, and autoimmune antigens.
 12. The method according to claim 11wherein the antigen is selected from the group consisting of influenzavirus, malaria, HIV, EBV, human papilloma virus, CMV, renal cellcarcinoma antigens, melanoma antigens, breast cancer antigens, cancerantigens and myeloma antigens.
 13. The method according to claim 11wherein the antigen is a tumor antigen.
 14. The method according toclaim 1 wherein said dendritic cells are immature and phagocytic. 15.The method according to claim 1 wherein the cells to be induced toundergo apoptosis are first transformed with DNA encoding said antigen.16. The method according to claim 1 wherein the ratio of apoptotic cellsto dendritic cells is about 1-10 apoptotic cells to about 100 dendriticcells.
 17. The method according to claim 1 further comprising amaturation step following internalization of said apoptotic cells bysaid dendritic cells wherein said dendritic cells are exposed to amaturation factor for a sufficient time to induce maturation of saiddendritic cells.
 18. The method according to claim 17 wherein thematuration step comprises contacting CD83 negative dendritic cells withat least one maturation factor selected from the group consisting ofmonocyte conditioned medium that causes CD83 negative dendritic cells tomature so as to express CD83, TNFα, IL-1β, IL-6, PGE₂, IFNα, CD40ligand, and necrotic cells.
 19. The method according to claim 18 whereinthe maturation factor is selected from the group consisting of monocyteconditioned medium; IFNα and at least one other factor selected from thegroup consisting of IL-1β, IL-6 and TNFα; and necrotic cells.
 20. Themethod according to claim 19 wherein the maturation factor is necroticcells.
 21. The method according to claim 1 wherein the antigen isproduced recombinantly.
 22. The method according to claim 21 whereinsaid apoptotic cells comprise said recombinantly produced antigen priorto becoming apoptotic.
 23. The method according to claim 1 wherein theantigen is a tumor antigen of cellular or viral origin.
 24. The methodof claim 1 wherein said dendritic cells are CD83 negative dendriticcells and maintaining the CD83 negative cells CD83 negative during saidcontacting.
 25. The method of claim 1 wherein the antigen is of viralorigin.
 26. The method of claim 1 wherein the antigen is an autoantigen.